Soluble CD40L (CD 154) as a prognostic marker of atherosclerotic diseases

ABSTRACT

This invention involves the new use of a diagnostic test to determine the risk of atherosclerotic diseases such as myocardial infarction and stroke, particularly among individuals with no signs or symptoms of current disease and among nonsmokers. Further, this invention involves the new use of a diagnostic test to assist physicians in determining which individuals at risk will preferentially benefit from certain treatments designed either to prevent first or recurrent myocardial infarctions and strokes, or to treat acute and chronic cardiovascular disorders. Methods for treatment are also described.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) from U.S.Provisional Application Ser. No. 60/338,841, filed Nov. 5, 2001, andentitled: SOLUBLE CD40L(CD154) AS A PROGNOSTIC MARKER OF ATHEROSCLEROTICDISEASES incorporated herein in its entirety by reference.

GOVERNMENT SUPPORT

The work leading to the present invention was funded in part by grantnumbers HL-34636, HL-56985, HL-58755, HL-63293 from the National Heart,Lung and Blood Institute. Accordingly, the United States Government mayhave certain rights to this invention.

FIELD OF THE INVENTION

This invention describes the new use of a diagnostic test to determinethe risk of cardiovascular disorders, such as myocardial infarction andstroke, particularly among individuals with no signs or symptoms ofcurrent disease and among nonsmokers. Further, this invention describesthe new use of a diagnostic test to assist physicians in determiningwhich individuals at risk will preferentially benefit from certaintreatments designed to prevent or treat cardiovascular disorders.Methods for treatment also are described.

BACKGROUND OF THE INVENTION

Despite significant advances in therapy, cardiovascular disease remainsthe single most common cause of morbidity and mortality in the developedworld. Thus, prevention of cardiovascular disorders such as myocardialinfarction and stroke is an area of major public health importance.Currently, several risk factors for future cardiovascular disorders havebeen described and are in wide clinical use in the detection ofindividuals at high risk. Such screening tests include evaluations oftotal and HDL cholesterol levels. However, a large number ofcardiovascular disorders occur in individuals with apparently low tomoderate risk profiles, and our ability to identify such patients islimited. Moreover, accumulating data suggests that the beneficialeffects of certain preventive and therapeutic treatments for patients atrisk for or known to have cardiovascular disorders differs in magnitudeamong different patient groups. At this time, however, data describingdiagnostic tests to determine whether certain therapies can be expectedto be more or less effective are lacking.

Certain cardiovascular disorders, such as myocardial infarction andischemic stroke, are associated with atherosclerosis. The mechanism ofatherosclerosis is not well understood. CD40 ligand (also known asCD40L, CD154, and/or gp39) is a 261 amino acid, type II transmembraneprotein. One or more biologically active soluble forms of the molecule,collectively designated sCD40L, are produced by proteolytic cleavage ofthe full-length form, which may occur intracellularly or on the cellsurface.

CD40L is a multipotent immunomodulator that together with its receptor,CD40, are expressed on a broad variety of cells including vascularendothelial (EC) and smooth muscle cells (SMC), mononuclear phagocytes(MO), and platelets.

Engagement of the CD40 receptor on any of the foregoing cell types,reportedly triggers the expression of various pro-inflammatorymediators, such as the cytokines IL1, IL-6, IL-12, TNFα, or IFNγ, thechemokines IL-8, MCP-1, or RANTES, the adhesion molecules ICAM-1 orVCAM-1, the matrix metalloproteinasesMMP-1/-2/-3/-7/-8/-9/-10/-11/-12/-13, as well as the procoagulant tissuefactor. Expression of these pro-inflammatory mediators has been,reportedly, linked to the promotion of a wide array of pro-atherogenicfunctions in vitro. These observations have implicated CD40L in thevarious stages of atherogenesis.

Elevated levels of sCD40L have been described among patients withunstable angina. Further, concentrations of sCD40L in serum or otherbody fluids have been used to assess the immune, inflammatory, ormalignant status of human patients. Such patients include patientssuffering from systemic autoimmunity or inflammation, vascular diseases,viral diseases, or malignancies, or patients undergoingimmunosuppressive therapy. These patients are not healthy individuals.Since levels of sCD40L increase during inflammation, it has beenuncertain whether statistical associations observed in these priorstudies of acutely ill or high-risk populations are causal, are due toshort-term inflammatory changes or are due to interrelations with otherrisk factors, in particular, smoking and hyperlipidemia.

Elevated levels of markers of inflammation have been shown previously tobe predictive of future adverse cardiovascular disorders. This has notpreviously been demonstrated for sCD40L, a mediator of certain aspectsof inflammation although not conventionally regarded previously as asystemic marker of inflammation.

SUMMARY OF THE INVENTION

This invention describes in one aspect new diagnostic tests whichbroadly include (1) the prediction of risk of future cardiovasculardisorders such as myocardial infarction and stroke and peripheralarterial disease; and (2) the determination of the likelihood thatcertain individuals will benefit to a greater or lesser extent from theuse of certain treatments designed to prevent and/or treatcardiovascular disorders. These new tests are based in part upon thefollowing discoveries.

It has been discovered that elevated levels of sCD40L are predictive offuture cardiovascular disorders. For example, elevated levels of sCD40Lin apparently healthy, nonsmokers are predictive of an increased risk ofmyocardial infarction. As another example, elevated levels of sCD40L arepredictive of an increased likelihood of a future stroke.

It has been discovered also that the likelihood that certain individualswill benefit to a greater or a lesser extent from the use of certaintherapeutic agents for reducing the risk of a future cardiovasculardisorder can be determined from the base-line level sCD40L in anindividual. The invention is based in part on the surprising discoverythat that sCD40L has a predictive value independent of other predictorsof future cardiovascular disorders. In particular, sCD40L predictsfuture adverse cardiovascular disorders independent of the systemicinflammatory marker C-Reactive Protein (CRP). Thus, sCD40L may be usedalone as a predictor future adverse cardiovascular disorders or incombination with prior art predictors such as cholesterol and CRP. Thus,the present invention does not involve simply duplicating a measurementthat previously could be made using other predictors. Instead, levels ofsCD40L are additive to prior art predictors.

As mentioned above, these discoveries have led to new diagnostic tests.

According to one aspect of the invention, a method is provided forcharacterizing an individual's risk profile of developing a futurecardiovascular disorder. The method involves obtaining a level of sCD40Lin the individual. The level of sCD40L then is compared to apredetermined value, and the individual's risk profile of developing afuture cardiovascular disorder then is characterized based upon thelevel of sCD40L in comparison to the predetermined value.

The predetermined value can be a single value, multiple values, a singlerange or multiple ranges. Thus, in one embodiment, the predeterminedvalue is a plurality of predetermined marker level ranges, and thecomparing step comprises determining in which of the predeterminedmarker level ranges the individual's level falls. In preferredembodiments, a preferred predetermined sCD40L value is about or above2.9 ng/mL of blood. Another preferred predetermined sCD40L value isabout or above 3.2 ng/mL of blood. A further preferred predeterminedsCD40L value is about or above 5.5 ng/mL of blood. When ranges areemployed, it is preferred that one of the plurality of ranges be belowabout 2.9 ng/mL of blood and that another of the ranges be above about2.9 ng/mL of blood.

In certain embodiments the individual is an apparently healthy,non-smoking individual. In some embodiments, the individual is nototherwise at an elevated risk of a myocardial infarction or stroke.

In some embodiments of this aspect of the invention, the cardiovasculardisorder is associated with atherosclerotic disease. In someembodiments, the cardiovascular disorder is other than fatal myocardialinfarction. In some embodiments, the cardiovascular disorder is astroke.

According to still another aspect of the invention, a method is providedfor characterizing an individual's risk profile of developing a futurecardiovascular disorder associated with atherosclerotic disease. A levelof sCD40L in the individual is obtained. The level of sCD40L is comparedto a predetermined value. The individual's risk profile of developingthe future cardiovascular disorder associated with atheroscleroticdisease, then is characterized based upon the level of sCD40L incomparison to the predetermined value. The predetermined value can be asdescribed above. The individual characterized may be any individual, butpreferably is an apparently healthy individual. The apparently healthyindividual can be a smoker or a nonsmoker. In certain embodiments thesubject does not otherwise have an elevated risk of an adversecardiovascular disorder. In certain embodiments, the futurecardiovascular disorder associated with atherosclerotic disease does notinclude a fatal myocardial infarction. The preferred markers andpredetermined values are as described above. In one importantembodiment, the cardiovascular disorder is stroke. In another importantembodiment, the cardiovascular disorder is myocardial infarction. Inanother important embodiment, the cardiovascular disorder is peripheralartery disease. In a further important embodiment, the cardiovasculardisorder is non-fatal myocardial infarction.

According to another aspect of the invention, a method is provided forcharacterizing an apparently healthy, non-smoking individual's riskprofile of developing a future myocardial infarction. The methodinvolves obtaining a level of sCD40L in the individual. The level ofsCD40L then is compared to a predetermined value, and the individual'srisk profile of developing a future myocardial infarction then ischaracterized based upon the level of sCD40L in comparison to thepredetermined value. In certain embodiments, the individual does nototherwise have an elevated risk of an adverse cardiovascular event.

As in the previous aspect of the invention, the predetermined value maybe a single value, a plurality of values, a single range or a pluralityof ranges. In one embodiment, the predetermined value is a plurality ofpredetermined marker level ranges and the comparing step involvesdetermining in which of the predetermined marker level ranges theindividual's level falls. Preferred predetermined values and the likefor sCD40L are as described above.

According to another aspect of the invention, a method is provided forcharacterizing an individual's risk profile of developing a futurecardiovascular disorder associated with atherosclerotic disease. A levelof sCD40L in the individual is obtained. The level of sCD40L is comparedto a predetermined value. The individual's risk profile of developingthe future cardiovascular disorder associated with atheroscleroticdisease, then is characterized based upon the level of sCD40L incomparison to the predetermined value. As in the previous aspect of theinvention, the predetermined value may be a single value, a plurality ofvalues, a single range or a plurality of ranges. In one embodiment, thepredetermined value is a plurality of predetermined marker level rangesand the comparing step involves determining in which of thepredetermined marker level ranges the individual's level falls.Preferred predetermined values and the like for sCD40L are as describedabove.

The individual characterized may be any individual, but preferably is anapparently healthy individual. The apparently healthy individual can bea smoker or a nonsmoker. In certain embodiments the subject does nototherwise have an elevated risk of an adverse cardiovascular event. Incertain embodiments, the future cardiovascular disorder associated withatherosclerotic disease does not include a fatal myocardial infarction.The preferred markers and predetermined values are as described above.In one important embodiment, the cardiovascular disorder is stroke. Inanother important embodiment, the cardiovascular disorder is myocardialinfarction. In another important embodiment, the cardiovascular disorderis peripheral artery disease. In a further important embodiment, thecardiovascular disorder is non-fatal myocardial infarction.

According to still another aspect of the invention, a method is providedin which one uses a blood sCD40L level together with a cholesterolfraction or C-Reactive Protein (CRP) for characterizing an individual'srisk profile of developing a future cardiovascular disorder associatedwith atherosclerotic disease. A level of sCD40L in the individual isobtained. The level of the sCD40L is compared to a first predeterminedvalue to establish a first risk value. A level of a cholesterol or CRPin the individual also is obtained. The level of the cholesterol or CRPin the individual is compared to a second predetermined value toestablish a second risk value. The individual's risk profile ofdeveloping the cardiovascular disorder then is characterized based uponthe combination of the first risk value and the second risk value,wherein the combination of the first risk value and second risk valueestablishes a third risk value different from the first and second riskvalues. In particularly important embodiments, the third risk value isgreater than either of the first and second risk values. The preferredindividuals for testing, markers and predetermined values are asdescribed above. The cardiovascular disorder can be any cardiovasculardisorder associated with atherosclerotic disease, although in certainimportant embodiments the cardiovascular disorder is nonfatal myocardialinfarction or ischemic stroke

According to yet another aspect of the invention, a method is providedfor evaluating the likelihood that an individual will benefit fromtreatment with an agent for reducing the risk of a cardiovasculardisorder, and particularly cardiovascular disorders associated withatherosclerotic disease. The agent can be selected from the groupconsisting of anti-inflammatory agents, anti-thrombotic agents,anti-platelet agents, fibrinolytic agents, lipid reducing agents, directthrombin inhibitors, and glycoprotein II b/IIIa receptor inhibitors andagents that bind to cellular adhesion molecules and inhibit the abilityof white blood cells to attach to such molecules (e.g. anti-cellularadhesion molecule antibodies). To practice the method, a level of sCD40Lin an individual is obtained. This level then is compared to apredetermined value, wherein the level of sCD40L in comparison to thepredetermined value is indicative of the likelihood that the individualwill benefit from treatment with the agent. The individual then can becharacterized in terms of the net benefit likely to be obtained bytreatment with the agent.

As mentioned above, the invention is particularly adapted to determiningwhich individuals will preferentially benefit from treatment with anagent for reducing the risk in the individuals of a cardiovasculardisorder such as a future stroke or a future myocardial infarction,including nonfatal myocardial infarctions. It also permits selection ofcandidate populations for clinical trials and for treatment withcandidate drugs, by identifying, for example, the individuals mostlikely to benefit from a new treatment or from a known treatment with ahigh risk profile of adverse side effects. Thus, the invention providesinformation for evaluating the likely net benefit of certain treatmentsfor candidate patients.

The invention also contemplates kits comprising a package including anassay for sCD40L and instructions, and optionally related materials suchas number or color charts, for correlating the level of sCD40L asdetermined by the assay with a risk of developing a futurecardiovascular disorder or with other patient criteria as describedabove. In important embodiments, the kits also include an assay for acholesterol.

In another aspect of the invention, a method for treating a subject toreduce the risk of a cardiovascular disorder, is provided. The methodinvolves selecting and administering to a subject who is known to havean above-normal level of sCD40L an agent for reducing the risk of thecardiovascular disorder. The agent can be an anti-inflammatory agent(including aspirin and nonaspirin anti-inflammatory agents), anantithrombotic agent, an anti-platelet agent, a fibrinolytic agent, alipid reducing agent, a direct thrombin inhibitor, a glycoproteinIIb/IIIa receptor inhibitor, an agent that binds to cellular adhesionmolecules and inhibits the ability of white blood cells to attach tosuch molecules, a calcium channel blocker, a beta-adrenergic receptorblocker, a cyclooxygenase-2 inhibitor, an angiotensin system inhibitor,and/or combinations thereof. The agent is administered in an amounteffective to lower the risk of the subject developing a futurecardiovascular disorder. The preferred subjects are apparently healthysubjects otherwise free of current need for treatment with any one orcombination of the foregoing agents. In further important embodiments,the subject treated is a nonhyperlipidemic subject. In anotherembodiment, the subjects are not at an elevated risk of an adversecardiovascular event (e.g., subjects with no family history of suchevents, subjects who are nonsmokers, subjects who are nonhyperlipidemicsubjects with normal levels of systemic inflammatory markers), otherthan having an elevated level of sCD40L.

In certain embodiments, the agent is an anti-inflammatory agent selectedfrom the group consisting of Alclofenac; Alclometasone Dipropionate;Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; AmfenacSodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen;Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; BenzydamineHydrochloride; Bromelains; Broperamole; Budesonide; Carprofen;Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; ClobetasoneButyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate;Cortodoxone; Deflazacort; Desonide; Desoximetasone; DexamethasoneDipropionate; Diclofenac Potassium; Diclofenac Sodium; DiflorasoneDiacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone;Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium;Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen;Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone;Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin;Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; HalopredoneAcetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol;Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen;Lofemizole Hydrochloride; Lornoxicam; Loteprednol Etabonate;Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate;Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate;Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone;Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone;Paranyline Hydrochloride; Pentosan Polysulfate Sodium; PhenbutazoneSodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; PiroxicamOlamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin;Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate;Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide;Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium;Triclonide; Triflumidate; Zidometacin; Glucocorticoids or ZomepiracSodium.

The invention also involves a method for treating subjects with a lipidreducing agent, to prevent cardiovascular disorders. Such an agent isadministered to a subject selected on the basis of having anabove-normal level of sCD40L. The agent is administered in an amounteffective to lower the risk of the subject developing a futurecardiovascular disorder. In one embodiment, the subject already has hada cardiovascular event, such as a heart attack or an angioplasty. Inthis embodiment, the lipid reducing agent can limit further injury orhelp prevent restenosis, post-myocardial infarction or post-angioplastyinjury. In another important embodiment, the subjects are apparentlyhealthy subjects otherwise free of current need for lipid reducing agenttreatment. In important embodiments, the subjects are not an elevatedrisk of an adverse cardiovascular event, other than having elevatedlevels of sCD40L. In further important embodiments, the subject treatedis a nonhyperlipidemic subject. In any of the foregoing embodiments, thelipid reducing agent may be, but is not limited to, gemfibrozil,cholystyramine, colestipol, nicotinic acid, probucol lovastatin,fluvastatin, simvastatin, atorvastatin, pravastatin, or cerivastatin. Inpreferred embodiments, the lipid reducing agent is pravastatin.

The invention also involves a method for treating subjects with an agentthat binds to a cellular adhesion molecule and that inhibits the abilityof white blood cells to attach to such molecules, to preventcardiovascular disorders. Such an agent is administered to a subjectselected on the basis of having an above-normal level of sCD40L. Theagent is administered in an amount effective to lower the risk of thesubject developing a future cardiovascular disorder. In one embodiment,the subject already has had a cardiovascular event, such as a heartattack or an angioplasty. In this embodiment, the agent that binds to acellular adhesion molecule and that inhibits the ability of white bloodcells to attach to such molecules, may limit further injury or helpprevent restenosis, post-myocardial infarction or post-angioplastyinjury. In another important embodiment, the subjects are apparentlyhealthy subjects otherwise free of current need for treatment with anagent that binds to a cellular adhesion molecule and that inhibits theability of white blood cells to attach to such molecules. In importantembodiments the subjects are not an elevated risk of an adversecardiovascular event, other than having elevated levels of sCD40L. Infurther important embodiments, the subject treated is anonhyperlipidemic subject.

The invention also involves a method for treating subjects with acalcium channel blocker, to prevent cardiovascular disorders. Such anagent is administered to a subject selected on the basis of having anabove-normal level of sCD40L. The agent is administered in an amounteffective to lower the risk of the subject developing a futurecardiovascular disorder. In one embodiment, the subject already has hada cardiovascular event, such as a heart attack or an angioplasty. Inanother important embodiment, the subjects are apparently healthysubjects otherwise free of current need for calcium channel blockertreatment. In important embodiments the subjects are not an elevatedrisk of an adverse cardiovascular event, other than having elevatedlevels of sCD40L. In further important embodiments, the subject treatedis a nonhyperlipidemic subject. In any of the foregoing embodiments, thecalcium channel blocker may be but is not limited to, dihydropyridines,phenyl alkyl amines, and/or benzothiazepines. In preferred embodiments,calcium channel blockers useful according to the invention, include, butare not limited to, amrinone, amlodipine, bencyclane, diltiazem,felodipine, fendiline, flunarizine, isradipine, nicardipine, nifedipine,nimodipine, perhexilene, gallopamil, tiapamil and tiapamil analogues(such as 1993RO-11-2933), verapamil, phenytoin, barbiturates, and thepeptides dynorphin, omega-conotoxin, and omega-agatoxin, and the likeand/or pharmaceutically acceptable salts thereof.

The invention also involves a method for treating subjects with abeta-adrenergic receptor blocker, to prevent cardiovascular disorders.Such an agent is administered to a subject selected on the basis ofhaving an above-normal level of sCD40L. The agent is administered in anamount effective to lower the risk of the subject developing a futurecardiovascular disorder. In one embodiment, the subject already has hada cardiovascular event, such as a heart attack or an angioplasty. Inanother important embodiment, the subjects are apparently healthysubjects otherwise free of current need for beta-adrenergic receptorblocker treatment. In important embodiments the subjects are not anelevated risk of an adverse cardiovascular event, other than havingelevated levels of sCD40L. In further important embodiments, the subjecttreated is a nonhyperlipidemic subject. In any of the foregoingembodiments, the beta-adrenergic receptor blocker may be but is notlimited to, atenolol, acebutolol, alprenolol, befunolol, betaxolol,bunitrolol, carteolol, celiprolol, hedroxalol, indenolol, labetalol,levobunolol, mepindolol, methypranol, metindol, metoprolol,metrizoranolol, oxprenolol, pindolol, propranolol, practolol, practolol,sotalolnadolol, tiprenolol, tomalolol, timolol, bupranolol, penbutolol,trimepranol,2-(3-(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl, 1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy) phthalide.

The invention also involves a method for treating subjects with acyclooxygenase-2 inhibitor, to prevent cardiovascular disorders. Such anagent is administered to a subject selected on the basis of having anabove-normal level of sCD40L. The agent is administered in an amounteffective to lower the risk of the subject developing a futurecardiovascular disorder. In one embodiment, the subject already has hada cardiovascular event, such as a heart attack or an angioplasty. Inanother important embodiment, the subjects are apparently healthysubjects otherwise free of current need for cyclooxygenase-2 inhibitortreatment. In important embodiments the subjects are not an elevatedrisk of an adverse cardiovascular event, other than having elevatedlevels of sCD40L. In further important embodiments, the subject treatedis a nonhyperlipidemic subject. In any of the foregoing embodiments, thecyclooxygenase-2 inhibitor may be, but is not limited to, a phenylheterocycle, a diaryl bicyclic heterocycle, an aryl substituted 5,5fused aromatic nitrogen compound, a N-benzylindol-3-yl propanoic acidand/or its derivatives, a 5-methanesulfonamido-1-indanone, a N-benzylindol-3-yl butanoic acid and/or its derivatives, adiphenyl-1,2-3-thiadiazole, a diaryl-5-oxygenated-2-(5H) -furanone, a3,4-diaryl-2-hydroxy-2,5-dihydrofuran, a stilbene and/or itsderivatives, a diphenyl stilbene, an alkylated styrene, a bisarylcyclobutene and/or its derivatives, a substituted pyridine, apyridinyl-2-cyclopenten-1-one, and/or a substitutedsulfonylphenylheterocycle.

The invention also involves a method for treating subjects with anangiotensin system inhibitor, to prevent cardiovascular disorders. Suchan agent is administered to a subject selected on the basis of having anabove-normal level of sCD40L. The agent is administered in an amounteffective to lower the risk of the subject developing a futurecardiovascular disorder. In one embodiment, the subject already has hada cardiovascular event, such as a heart attack or an angioplasty. Inanother important embodiment, the subjects are apparently healthysubjects otherwise free of current need for angiotensin system inhibitortreatment. In important embodiments the subjects are not an elevatedrisk of an adverse cardiovascular event, other than having elevatedlevels of sCD40L. In further important embodiments, the subject treatedis a nonhyperlipidemic subject. In any of the foregoing embodiments, theangiotensin system inhibitor may be, but is not limited to, anangiotensin-converting enzyme (ACE) inhibitor, an angiotensin IIantagonist, an angiotensin II receptor antagonist, agents that activatethe catabolism of angiotensin II, and/or agents that prevent thesynthesis of angiotensin I.

According to another aspect of the invention, a method is provided forevaluating the likelihood that an individual will benefit from treatmentwith an agent for reducing the risk of a cardiovascular disorderassociated with atherosclerotic disease. The agent can be selected fromthe group consisting of anti-inflammatory agents, anti-thromboticagents, anti-platelet agents, fibrinolytic agents, lipid reducingagents, direct thrombin inhibitors, glycoprotein II b/IIIa receptorinhibitors, agents that bind to cellular adhesion molecules and inhibitthe ability of white blood cells to attach to such molecules (e.g.anti-cellular adhesion molecule antibodies), calcium channel blockers,beta-adrenergic receptor blockers, cyclooxygenase-2 inhibitors,angiotensin system inhibitors, and/or combinations of the foregoingagents thereof. To practice the method, a level of sCD40L in anindividual is obtained. This level then is compared to a predeterminedvalue, wherein the level of sCD40L in comparison to the predeterminedvalue is indicative of the likelihood that the individual will benefitfrom treatment with the agent. The individual then can be characterizedin terms of the net benefit likely to be obtained by treatment with theagent.

The predetermined value can be as described above.

As mentioned above, the invention is particularly adapted to determiningwhich individuals will preferentially benefit from treatment with anagent for reducing the risk in the individuals of a cardiovasculardisorder such as a future stroke or a future myocardial infarction,including nonfatal myocardial infarctions. It also permits selection ofcandidate populations for clinical trials and for treatment withcandidate drugs, by identifying, for example, the individuals mostlikely to benefit from a new treatment or from a known treatment with ahigh risk profile of adverse side effects. Thus, the invention providesinformation for evaluating the likely net benefit of certain treatmentsfor candidate patients.

According to another aspect of the invention, a method for reducingsCD40L levels in a subject to lower the risk of an adversecardiovascular disorder is provided. The method involves selecting andadministering to a subject having elevated levels of sCD40L an agentthat reduces sCD40L levels in an amount effective to reduce the sCD40Llevels in the subject. In one embodiment, the agent is a lipid reducingagent. The preferred subject is an apparently healthy subject. In someembodiments, the subject is not otherwise at an elevated risk of havingan adverse cardiovascular event. In certain embodiments, the subject haselevated C-Reactive Protein (CRP) levels. In some embodiments of thisaspect of the invention, the subject is otherwise free of indicationscalling for treatment with a lipid reducing agent.

According to a further aspect of the invention, a method for evaluatingthe likelihood for vascular intra-plaque lipid accumulation in anindividual at risk of developing a cardiovascular disorder, is provided.The method involves obtaining a level of sCD40L in the individual,comparing the level of sCD40L to a predetermined value, andcharacterizing the individual's risk profile for vascular intra-plaquelipid accumulation, based upon the level of sCD40L in comparison to thepredetermined level. The predetermined value can be a plurality ofpredetermined sCD40L level ranges and said comparing step comprisesdetermining in which of said predetermined sCD40L level ranges saidindividual's sCD40L level falls. In certain embodiments, thepredetermined value is about 2.5 ng/mL of blood or higher. In someembodiments, the predetermined value is about 3.0 ng/mL of blood orhigher. In important embodiments, the predetermined value is a pluralityof predetermined sCD40L level ranges, one of said plurality being belowabout 2.5 ng/mL blood and another of said ranges being about 2.5 ng/mLblood, and said comparing step comprises determining in which of saidplurality of predetermined sCD40L level ranges said individual's sCD40Llevel falls. In further important embodiments, the vascular intra-plaquelipid accumulation occurs in the carotid artery. Other preferred rangesand important embodiments are as described above and below.

According to a further aspect of the invention, methods for preparingmedicaments useful in the treatment of cardiovascular conditions, areprovided.

These and other aspects of the invention will be described in moredetail below in connection with the detailed description of theinvention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph demonstrating the relative risk of cardiovascularevents in the study population according to sCD40L blood levels. Thedotted line depicts the 99^(th) percentile cutpoint for the controldistribution.

DETAILED DESCRIPTION OF THE INVENTION

The primary basis for this invention is evidence from a prospective,nested case control analysis among participants in the Women's HealthStudy (WHS), an ongoing primary prevention trial evaluating theefficiency of vitamin E and low dose aspirin in 28,263 middle agedAmerican women with no history of cardiovascular disease or cancer. Inthis trial, baseline level of sCD40L, a mediator of inflammation, wasfound to determine the future risk of myocardial infarction and stroke,independent of a large series of lipid and non-lipid risk factors, andindependent of other predictors, including markers of systemicinflammation (see, e.g., U.S. Pat. No. 6,040,147). Specifically,individuals with the highest baseline levels of sCD40L were found tohave at least 3 fold increases in risk of developing futurecardiovascular events (FIG. 1).

Moreover, in data from the Women's Health Study, the risk of futuremyocardial infarction and stroke associated with sCD40L, appear to beadditive to that which could otherwise be determined by usual assessmentof total cholesterol and HDL cholesterol. In this trial, the predictivevalue of sCD40L was present for non-fatal as well as fatal events, wasstable over long periods of time, and was present for non-smokers aswell as smokers. Further, data from this trial indicate the magnitude ofbenefit that apparently healthy individuals can expect from therapeuticagents used in the prevention and treatment of atheroscleroticdisorders.

The current invention in one aspect describes the use of sCD40L topredict risk of cardiovascular disorders associated with atherosclerosissuch as myocardial infarction and stroke among individuals withoutcurrent evidence of disease. Thus, these data greatly extend priorobservations regarding the use of sCD40L, to predict risk among alreadyidentified high-risk populations or among symptomatic ischemia patientssuch as those with unstable angina pectoris. Indeed, since levels ofsCD40L increase following acute ischemia, it has been uncertain whetherstatistical associations observed in prior studies of acutely ill orhigh-risk populations are casual or due to short-term inflammatorychanges, or to interrelations with other risk factors, in particularsmoking and hyperlipidemia.

In marked contrast, data from the Women's Health Study indicate for thefirst time the utility of sCD40L to predict risk among currently healthyand otherwise low-risk individuals, to predict non-fatal as well asfatal events, to predict risk among non-smokers, and to predict riskabove and beyond that associated with screening for total and HDLcholesterol. Data from the Women's Health Study also indicate for thefirst time that the likelihood of efficacy of interventions designed toreduce risk of atherosclerotic events such as myocardial infarction andstroke differs in magnitude based upon a measure of the sCD40Lplasma/blood levels. The invention will be better understood withreference to the following brief explanation of terms.

“Cardiovascular myocardial ischemia, disorders” includes myocardialinfarction, stroke, myocardial ischemia, angina pectoris and peripheralarteriovascular disease. Cardiovascular disorders do not include venousthrombosis.

“Apparently healthy”, as used herein, means individuals who have notpreviously had an acute adverse cardiovascular event such as amyocardial infarction (i.e., individuals who are not at an elevated riskof a second adverse cardiovascular event due to a primary adversecardiovascular event). Apparently healthy individuals also do nototherwise exhibit symptoms of disease. In other words, such individuals,if examined by a medical professional, would be characterized as healthyand free of symptoms of disease.

In important embodiments, the subject does not otherwise have anelevated risk of an adverse cardiovascular event. Subjects having anelevated such risk include those with a family history of cardiovasculardisease, elevated lipids, smokers, prior acute cardiovascular event,etc. (See, e.g., Harrison's Principles of Experimental Medicine, 15thEdition, McGraw-Hill, Inc., N.Y.—hereinafter “Harrison's”). According toone important aspect of the invention, a method for treating a subjectto reduce the risk of a cardiovascular disorder, is provided. The methodinvolves selecting and administering to a subject who is known to havean above-normal level of sCD40L an agent for reducing the risk of thecardiovascular disorder. The agent can be an anti-inflammatory agent, anantithrombotic agent, an anti-platelet agent, a fibrinolytic agent, alipid reducing agent, a direct thrombin inhibitor, a glycoproteinIIb/IIIa receptor inhibitor, an agent that binds to cellular adhesionmolecules and inhibits the ability of white blood cells to attach tosuch molecules, a calcium channel blocker, a beta-adrenergic receptorblocker, a cyclooxygenase-2 inhibitor, an angiotensin system inhibitor,and/or combinations thereof. The agent is administered in an amounteffective to lower the risk of the subject developing a futurecardiovascular disorder.

The preferred subjects are apparently healthy subjects otherwise free ofcurrent need for treatment with the agent prescribed according to thepresent invention. For example, if treatment with a particular agentoccurs based on elevated levels of sCD40L, then the patient preferablyis free of symptoms calling for treatment with that agent (or thecategory of agent into which the agent falls), other than the symptom ofhaving elevated levels of sCD40L. In some embodiments, the subject isotherwise free of symptoms calling for treatment with any one of anycombination of or all of the foregoing categories of agents. Such as,for example, with respect to anti-inflammatory agents, free of symptomsof rheumatoid arthritis, chronic back pain, autoimmune diseases,vascular diseases, viral diseases, malignancies, and the like. Infurther important embodiments, the subject treated is anonhyperlipidemic subject. In another embodiment, the subjects are notat an elevated risk of an adverse cardiovascular event (e.g., subjectswith no family history of such events, subjects who are nonsmokers,subjects who are nonhyperlipidemic, subjects who do not have elevatedlevels of a systemic inflammatory marker), other than having an elevatedlevel of sCD40L. In some embodiments, the subject is otherwise free ofsymptoms calling for treatment with any one of, any combination of orall of the foregoing categories of agents.

In some embodiments, the subject is otherwise free of symptoms callingfor treatment with any one of, any combination of or all of theforegoing categories of agents. In further important embodiments, thesubject treated is a nonhyperlipidemic subject. A “nonhyperlipidemic” isa subject that is a nonhypercholesterolemic and/or anonhypertriglyceridemic subject. A “nonhypercholesterolemic” subject isone that does not fit the current criteria established for ahypercholesterolemic subject. A nonhypertriglyceridemic subject is onethat does not fit the current criteria established for ahypertriglyceridemic subject (See, e.g., Harrison's Principles ofExperimental Medicine, 15th Edition, McGraw-Hill, Inc., N.Y.—hereinafter“Harrison's”). Hypercholesterolemic subjects and hypertriglyceridemicsubjects are associated with increased incidence of premature coronaryheart disease. A hypercholesterolemic subject has an LDL level of >160mg/dL, or >130 mg/dL and at least two risk factors selected from thegroup consisting of male gender, family history of premature coronaryheart disease, cigarette smoking (more than 10 per day), hypertension,low HDL (<35 mg/dL), diabetes mellitus, hyperinsulinemia, abdominalobesity, high lipoprotein (a), and personal history of cerebrovasculardisease or occlusive peripheral vascular disease. A hypertriglyceridemicsubject has a triglyceride (TG) level of >250 mg/dL. Thus, anonhyperlipidemic subject is defined as one whose cholesterol andtriglyceride levels are below the limits set as described above for boththe hypercholesterolemic and hypertriglyceridemic subjects.

In some embodiments, the subject has normal levels of systemicinflammatory markers. For the purposes of this application, normal suchlevels means the same things as the absence of elevated levels. Normallevels will depend on the particular systemic inflammatory marker. (SeeU.S. Pat. No. 6,040,147, incorporated herein in its entirety byreference.)

“Nonsmoking”, as used herein, means an individual who, at the time ofthe evaluation, is not a smoker. This includes individuals who havenever smoked as well as individuals who in the past have smoked butpresently no longer smoke.

Agents for reducing the risk of a cardiovascular disorder include, butare not limited to, those selected from the group consisting ofanti-inflammatory agents, anti-thrombotic agents, anti-platelet agents,fibrinolytic agents, lipid reducing agents, direct thrombin inhibitors,glycoprotein II b/IIIa receptor inhibitors, agents that bind to cellularadhesion molecules and inhibit the ability of white blood cells toattach to such molecules (e.g. anti-cellular adhesion moleculeantibodies), calcium channel blockers, beta-adrenergic receptorblockers, cyclooxygenase-2 inhibitors, angiotensin system inhibitors,and/or any combinations thereof.

“Anti-inflammatory” agents include but are not limited to, Alclofenac;Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase;Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride;Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium;Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;Clobetasol Propionate; Clobetasone Butyrate; Clopirac; CloticasonePropionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium;Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone;Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen;Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; ProxazoleCitrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate;Salycilates; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam;Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone;Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine;Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide;Triflumidate; Zidometacin; Glucocorticoids; Zomepirac Sodium.

“Anti-thrombotic” and/or “fibrinolytic” agents include but are notlimited to, Plasminogen (to plasmin via interactions of prekallikrein,kininogens, Factors XII, XIIIa, plasminogen proactivator, and tissueplasminogen activator[TPA]) Streptokinase; Urokinase: AnisoylatedPlasminogen-Streptokinase Activator Complex; Pro-Urokinase; (Pro-UK);rTPA (alteplase or activase; r denotes recombinant); rPro-UK;Abbokinase; Eminase; Sreptase Anagrelide Hydrochloride; Bivalirudin;Dalteparin Sodium; Danaparoid Sodium; Dazoxiben Hydrochloride; EfegatranSulfate; Enoxaparin Sodium; Ifetroban; Ifetroban Sodium; TinzaparinSodium; retaplase; Trifenagrel; Warfarin; Dextrans.

“Anti-platelet” agents include but are not limited to, Clopridogrel;Sulfinpyrazone; Aspirin; Dipyridamole; Clofibrate; Pyridinol Carbamate;PGE; Glucagon; Antiserotonin drugs; Caffeine; Theophyllin Pentoxifyllin;Ticlopidine; Anagrelide.

“Lipid reducing” agents include but are not limited to, gemfibrozil,cholystyramine, colestipol, nicotinic acid, probucol lovastatin,fluvastatin, simvastatin, atorvastatin, pravastatin, cerivastatin, andother HMG-CoA reductase inhibitors.

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is themicrosomal enzyme that catalyzes the rate limiting reaction incholesterol biosynthesis (HMG-CoA6Mevalonate). An HMG-CoA reductaseinhibitor inhibits HMG-CoA reductase, and as a result inhibits thesynthesis of cholesterol. A number of HMG-CoA reductase inhibitors hasbeen used to treat individuals with hypercholesterolemia. More recently,HMG-CoA reductase inhibitors have been shown to be beneficial in thetreatment of stroke (Endres M, et al., Proc Natl Acad Sci USA, 1998,95:8880-5).

HMG-CoA reductase inhibitors useful according to the invention include,but are not limited to, simvastatin (U.S. Pat. No. 4,444,784),lovastatin (U.S. Pat. No. 4,231,938), pravastatin sodium (U.S. Pat. No.4,346,227), fluvastatin (U.S. Pat. No. 4,739,073), atorvastatin (U.S.Pat. No. 5,273,995), cerivastatin, and numerous others described in U.S.Pat. No. 5,622,985, U.S. Pat. No. 5,135,935, U.S. Pat. No. 5,356,896,U.S. Pat. No. 4,920,109, U.S. Pat. No. 5,286,895, U.S. Pat. No.5,262,435, U.S. Pat. No. 5,260,332, U.S. Pat. No. 5,317,031, U.S. Pat.No. 5,283,256, U.S. Pat. No. 5,256,689, U.S. Pat. No. 5,182,298, U.S.Pat. No. 5,369,125, U.S. Pat. No. 5,302,604, U.S. Pat. No. 5,166,171,U.S. Pat. No. 5,202,327, U.S. Pat. No. 5,276,021, U.S. Pat. No.5,196,440, U.S. Pat. No. 5,091,386, U.S. Pat. No. 5,091,378, U.S. Pat.No. 4,904,646, U.S. Pat. No. 5,385,932, U.S. Pat. No. 5,250,435, U.S.Pat. No. 5,132,312, U.S. Pat. No. 5,130,306, U.S. Pat. No. 5,116,870,U.S. Pat. No. 5,112,857, U.S. Pat. No. 5,102,911, U.S. Pat. No.5,098,931, U.S. Pat. No. 5,081,136, U.S. Pat. No. 5,025,000, U.S. Pat.No. 5,021,453, U.S. Pat. No. 5,017,716, U.S. Pat. No. 5,001,144, U.S.Pat. No. 5,001,128, U.S. Pat. No. 4,997,837, U.S. Pat. No. 4,996,234,U.S. Pat. No. 4,994,494, U.S. Pat. No. 4,992,429, U.S. Pat. No.4,970,231, U.S. Pat. No. 4,968,693, U.S. Pat. No. 4,963,538, U.S. Pat.No. 4,957,940, U.S. Pat. No. 4,950,675, U.S. Pat. No. 4,946,864, U.S.Pat. No. 4,946,860, U.S. Pat. No. 4,940,800, U.S. Pat. No. 4,940,727,U.S. Pat. No. 4,939,143, U.S. Pat. No. 4,929,620, U.S. Pat. No.4,923,861, U.S. Pat. No. 4,906,657, U.S. Pat. No. 4,906,624 and U.S.Pat. No. 4,897,402, the disclosures of which patents are incorporatedherein by reference.

“Direct thrombin inhibitors” include but are not limited to, hirudin,hirugen, hirulog, agatroban, PPACK, thrombin aptamers.

“Glycoprotein IIb/IIIa receptor inhibitors” are both antibodies andnon-antibodies, and include but are not limited to ReoPro (abcixamab),lamifiban, tirofiban.

“Calcium channel blockers” are a chemically diverse class of compoundshaving important therapeutic value in the control of a variety ofdiseases including several cardiovascular disorders, such ashypertension, angina, and cardiac arrhythmias (Fleckenstein, Cir. Res.v. 52, (suppl. 1), p.13-16 (1983); Fleckenstein, Experimental Facts andTherapeutic Prospects, John Wiley, New York (1983); McCall, D., CurrPract Cardiol, v. 10, p. 1-11 (1985)). Calcium channel blockers are aheterogenous group of drugs that prevent or slow the entry of calciuminto cells by regulating cellular calcium channels. (Remington, TheScience and Practice of Pharmacy, Nineteenth Edition, Mack PublishingCompany, Eaton, Pa., p. 963 (1995)). Most of the currently availablecalcium channel blockers, and useful according to the present invention,belong to one of three major chemical groups of drugs, thedihydropyridines, such as nifedipine, the phenyl alkyl amines, such asverapamil, and the benzothiazepines, such as diltiazem. Other calciumchannel blockers useful according to the invention, include, but are notlimited to, amrinone, amlodipine, bencyclane, felodipine, fendiline,flunarizine, isradipine, nicardipine, nimodipine, perhexilene,gallopamil, tiapamil and tiapamil analogues (such as 1993RO-11-2933),phenytoin, barbiturates, and the peptides dynorphin, omega-conotoxin,and omega-agatoxin, and the like and/or pharmaceutically acceptablesalts thereof.

“Beta-adrenergic receptor blocking agents” are a class of drugs thatantagonize the cardiovascular effects of catecholamines in anginapectoris, hypertension, and cardiac arrhythmias. Beta-adrenergicreceptor blockers include, but are not limited to, atenolol, acebutolol,alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol,hedroxalol, indenolol, labetalol, levobunolol, mepindolol, methypranol,metindol, metoprolol, metrizoranolol, oxprenolol, pindolol, propranolol,practolol, practolol, sotalolnadolol, tiprenolol, tomalolol, timolol,bupranolol, penbutolol, trimepranol,2-(3-(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl,1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy)phthalide.The above-identified compounds can be used as isomeric mixtures, or intheir respective levorotating or dextrorotating form.

Cyclooxygenase-2 (COX-2) is a recently identified new form of acyclooxygenase. “Cyclooxygenase” is an enzyme complex present in mosttissues that produces various prostaglandins and thromboxanes fromarachidonic acid. Non-steroidal, antiinflammatory drugs exert most oftheir antiinflammatory, analgesic and antipyretic activity and inhibithormone-induced uterine contractions and certain types of cancer growththrough inhibition of the cyclooxygenase (also known as prostaglandinG/H synthase and/or prostaglandin-endoperoxide synthase). Initially,only one form of cyclooxygenase was known, the “constitutive enzyme” orcyclooxygenase-1 (COX-1). It and was originally identified in bovineseminal vesicles.

Cyclooxygenase-2 (COX-2) has been cloned, sequenced and characterizedinitially from chicken, murine and human sources (See, e.g., U.S. Pat.No. 5,543,297, issued Aug. 6, 1996 to Cromlish, et al., and assigned toMerck Frosst Canada, Inc., Kirkland, Calif., entitled: “Humancyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity”). This enzyme is distinct from the COX-1. COX-2, is rapidlyand readily inducible by a number of agents including mitogens,endotoxin, hormones, cytokines and growth factors. As prostaglandinshave both physiological and pathological roles, it is believed that theconstitutive enzyme, COX-1, is responsible, in large part, forendogenous basal release of prostaglandins and hence is important intheir physiological functions such as the maintenance ofgastrointestinal integrity and renal blood flow. By contrast, it isbelieved that the inducible form, COX-2, is mainly responsible for thepathological effects of prostaglandins where rapid induction of theenzyme would occur in response to such agents as inflammatory agents,hormones, growth factors, and cytokines. Therefore, it is believed thata selective inhibitor of COX-2 has similar antiinflammatory, antipyreticand analgesic properties to a conventional non-steroidalantiinflammatory drug, and in addition inhibits hormone-induced uterinecontractions and also has potential anti-cancer effects, but withreduced side effects. In particular, such COX-2 inhibitors are believedto have a reduced potential for gastrointestinal toxicity, a reducedpotential for renal side effects, a reduced effect on bleeding times andpossibly a decreased potential to induce asthma attacks inaspirin-sensitive asthmatic subjects, and are therefore useful accordingto the present invention.

A number of selective “COX-2 inhibitors” are known in the art. Theseinclude, but are not limited to, COX-2 inhibitors described in U.S. Pat.No. 5,474,995 “Phenyl heterocycles as cox-2 inhibitors”; U.S. Pat. No.5,521,213 “Diaryl bicyclic heterocycles as inhibitors ofcyclooxygenase-2″; U.S. Pat. No. 5,536,752 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,550,142 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,552,422 “Aryl substituted 5,5 fusedaromatic nitrogen compounds as anti-inflammatory agents”; U.S. Pat. No.5,604,253 “N-benzylindol-3-yl propanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,604,260“5-methanesulfonamido-1-indanones as an inhibitor of cyclooxygenase-2”;U.S. Pat. No. 5,639,780 N-benzyl indol-3-yl butanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,677,318Diphenyl-1,2-3-thiadiazoles as anti-inflammatory agents”; U.S. Pat. No.5,691,374 “Diaryl-5-oxygenated-2-(5H) -furanones as COX-2 inhibitors”;U.S. Pat. No. 5,698,584 “3,4-diaryl-2-hydroxy-2,5-dihydrofurans asprodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,710,140 “Phenylheterocycles as COX-2 inhibitors”; U.S. Pat. No. 5,733,909 “Diphenylstilbenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,789,413“Alkylated styrenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No.5,817,700 “Bisaryl cyclobutenes derivatives as cyclooxygenaseinhibitors”; U.S. Pat. No. 5,849,943 “Stilbene derivatives useful ascyclooxygenase-2 inhibitors”; U.S. Pat. No. 5,861,419 “Substitutedpyridines as selective cyclooxygenase-2 inhibitors”; U.S. Pat. No.5,922,742 “Pyridinyl-2-cyclopenten-1-ones as selective cyclooxygenase-2inhibitors”; U.S. Pat. No. 5,925,631 “Alkylated styrenes as prodrugs toCOX-2 inhibitors”; all of which are commonly assigned to Merck FrosstCanada, Inc. (Kirkland, Calif.). Additional COX-2 inhibitors are alsodescribed in U.S. Pat. No. 5,643,933, assigned to G. D. Searle & Co.(Skokie, Ill.), entitled: “Substituted sulfonylphenylheterocycles ascyclooxygenase-2 and 5-lipoxygenase inhibitors.”

A number of the above-identified COX-2 inhibitors are prodrugs ofselective COX-2 inhibitors, and exert their action by conversion in vivoto the active and selective COX-2 inhibitors. The active and selectiveCOX-2 inhibitors formed from the above-identified COX-2 inhibitorprodrugs are described in detail in WO 95/00501, published Jan. 5, 1995,WO 95/18799, published Jul. 13, 1995 and U.S. Pat. No. 5,474,995, issuedDec. 12, 1995. Given the teachings of U.S. Pat. No. 5,543,297, entitled:“Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity,” a person of ordinary skill in the art would be able todetermine whether an agent is a selective COX-2 inhibitor or a precursorof a COX-2 inhibitor, and therefore part of the present invention.

An “angiotensin system inhibitor” is an agent that interferes with thefunction, synthesis or catabolism of angiotensin II. These agentsinclude, but are not limited to, angiotensin-converting enzyme (ACE)inhibitors, angiotensin II antagonists, angiotensin II receptorantagonists, agents that activate the catabolism of angiotensin II, andagents that prevent the synthesis of angiotensin I from whichangiotensin II is ultimately derived. The renin-angiotensin system isinvolved in the regulation of hemodynamics and water and electrolytebalance. Factors that lower blood volume, renal perfusion pressure, orthe concentration of Na⁺ in plasma tend to activate the system, whilefactors that increase these parameters tend to suppress its function.

Angiotensin I and angiotensin II are synthesized by the enzymaticrenin-angiotensin pathway. The synthetic process is initiated when theenzyme renin acts on angiotensinogen, a pseudoglobulin in blood plasma,to produce the decapeptide angiotensin I. Angiotensin I is converted byangiotensin converting enzyme (ACE) to angiotensin II (angiotensin-[1-8]octapeptide). The latter is an active pressor substance which has beenimplicated as a causative agent in several forms of hypertension invarious mammalian species, e.g., humans.

Angiotensin (renin-angiotensin) system inhibitors are compounds that actto interfere with the production of angiotensin II from angiotensinogenor angiotensin I or interfere with the activity of angiotensin II. Suchinhibitors are well known to those of ordinary skill in the art andinclude compounds that act to inhibit the enzymes involved in theultimate production of angiotensin II, including renin and ACE. Theyalso include compounds that interfere with the activity of angiotensinII, once produced. Examples of classes of such compounds includeantibodies (e.g., to renin), amino acids and analogs thereof (includingthose conjugated to larger molecules), peptides (including peptideanalogs of angiotensin and angiotensin I), pro-renin related analogs,etc. Among the most potent and useful renin-angiotensin systeminhibitors are renin inhibitors, ACE inhibitors, and angiotensin IIantagonists. In a preferred embodiment of the invention, therenin-angiotensin system inhibitors are renin inhibitors, ACEinhibitors, and angiotensin II antagonists.

“Angiotensin II antagonists” are compounds which interfere with theactivity of angiotensin II by binding to angiotensin II receptors andinterfering with its activity. Angiotensin II antagonists are well knownand include peptide compounds and non-peptide compounds. Mostangiotensin II antagonists are slightly modified congeners in whichagonist activity is attenuated by replacement of phenylalanine inposition 8 with some other amino acid; stability can be enhanced byother replacements that slow degeneration in vivo. Examples ofangiotensin II antagonists include: peptidic compounds (e.g., saralasin,[(San¹⁾(Val⁵)(Ala⁸)] angiotensin -(1-8) octapeptide and relatedanalogs); N-substituted imidazole-2-one (U.S. Pat. No. 5,087,634);imidazole acetate derivatives including2-N-butyl-4-chloro-1-(2-chlorobenzile) imidazole-5-acetic acid (see Longet al., J. Pharmacol. Exp. Ther. 247(1), 1-7 (1988));4,5,6,7-tetrahydro-1H-imidazo [4,5-c] pyridine-6-carboxylic acid andanalog derivatives (U.S. Pat. No. 4,816,463); N2-tetrazolebeta-glucuronide analogs (U.S. Pat. No. 5,085,992); substitutedpyrroles, pyrazoles, and tryazoles (U.S. Pat. No. 5,081,127); phenol andheterocyclic derivatives such as 1, 3-imidazoles (U.S. Pat. No.5,073,566); imidazo-fused 7-member ring heterocycles (U.S. Pat. No.5,064,825); peptides (e.g., U.S. Pat. No. 4,772,684); antibodies toangiotensin II (e.g., U.S. Pat. No. 4,302,386); and aralkyl imidazolecompounds such as biphenyl-methyl substituted imidazoles (e.g., EPNumber 253,310, Jan. 20, 1988); ES8891(N-morpholinoacetyl-(-1-naphthyl)-L-alanyl-(4, thiazolyl)-L-alanyl(35,45)-4-amino-3-hydroxy-5-cyclo-hexapentanoyl-N-hexylamide, SankyoCompany, Ltd., Tokyo, Japan); SKF108566 (E-alpha-2-[2-butyl-1-(carboxyphenyl) methyl] 1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid,Smith Kline Beecham Pharmaceuticals, PA); Losartan (DUP753/MK954, DuPontMerck Pharmaceutical Company); Remikirin (RO42-5892, F. Hoffman LaRocheAG); A₂ agonists (Marion Merrill Dow) and certain non-peptideheterocycles (G.D. Searle and Company).

“Angiotensin converting enzyme (ACE), is an enzyme which catalyzes theconversion of angiotensin I to angiotensin II. ACE inhibitors includeamino acids and derivatives thereof, peptides, including di and tripeptides and antibodies to ACE which intervene in the renin-angiotensinsystem by inhibiting the activity of ACE thereby reducing or eliminatingthe formation of pressor substance angiotensin II. ACE inhibitors havebeen used medically to treat hypertension, congestive heart failure,myocardial infarction and renal disease. Classes of compounds known tobe useful as ACE inhibitors include acylmercapto and mercaptoalkanoylprolines such as captopril (U.S. Pat. No. 4,105,776) and zofenopril(U.S. Pat. No. 4,316,906), carboxyalkyl dipeptides such as enalapril(U.S. Pat. No. 4,374,829), lisinopril (U.S. Pat. No. 4,374,829),quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S. Pat. No. 4,587,258),and perindopril (U.S. Pat. No. 4,508,729), carboxyalkyl dipeptide mimicssuch as cilazapril (U.S. Pat. No. 4,512,924) and benazapril (U.S. Pat.No. 4,410,520), phosphinylalkanoyl prolines such as fosinopril (U.S.Pat. No. 4,337,201) and trandolopril.

“Renin inhibitors” are compounds which interfere with the activity ofrenin. Renin inhibitors include amino acids and derivatives thereof,peptides and derivatives thereof, and antibodies to renin. Examples ofrenin inhibitors that are the subject of United States patents are asfollows: urea derivatives of peptides (U.S. Pat. No. 5,116,835); aminoacids connected by nonpeptide bonds (U.S. Pat. No. 5,114,937); di andtri peptide derivatives (U.S. Pat. No. 5,106,835); amino acids andderivatives thereof (U.S. Pat. Nos. 5,104,869 and 5,095,119); diolsulfonamides and sulfinyls (U.S. Pat. No. 5,098,924); modified peptides(U.S. Pat. No. 5,095,006); peptidyl beta-aminoacyl aminodiol carbamates(U.S. Pat. No. 5,089,471); pyrolimidazolones (U.S. Pat. No. 5,075,451);fluorine and chlorine statine or statone containing peptides (U.S. Pat.No. 5,066,643); peptidyl amino diols (U.S. Pat. Nos. 5,063,208 and4,845,079); N-morpholino derivatives (U.S. Pat. No. 5,055,466);pepstatin derivatives (U.S. Pat. No. 4,980,283); N-heterocyclic alcohols(U.S. Pat. No. 4,885,292); monoclonal antibodies to renin (U.S. Pat. No.4,780,401); and a variety of other peptides and analogs thereof (U.S.Pat. Nos. 5,071,837, 5,064,965, 5,063,207, 5,036,054, 5,036,053,5,034,512, and 4,894,437).

Agents that bind to cellular adhesion molecules and inhibit the abilityof white blood cells to attach to such molecules include polypeptideagents. Such polypeptides include polyclonal and monoclonal antibodies,prepared according to conventional methodology. Such antibodies alreadyare known in the art and include anti-ICAM 1 antibodies as well as othersuch antibodies. Significantly, as is well-known in the art, only asmall portion of an antibody molecule, the paratrope, is involved in thebinding of the antibody to its epitope (see, in general, Clark, W. R.(1986) The Experimental Foundations of Modern Immunology, Wiley & Sons,Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed.,Blackwell Scientific Publications, Oxford). The pFc′ and Fc regions, forexample, are effectors of the complement cascade but are not involved inantigen binding. An antibody from which the pFc′ region has beenenzymatically cleaved, or which has been produced without the pFc′region, designated an F(ab′)₂ fragment, retains both of the antigenbinding sites of an intact antibody. Similarly, an antibody from whichthe Fc region has been enzymatically cleaved, or which has been producedwithout the Fc region, designated an Fab fragment, retains one of theantigen binding sites of an intact antibody molecule. Proceedingfurther, Fab fragments consist of a covalently bound antibody lightchain and a portion of the antibody heavy chain denoted Fd. The Fdfragments are the major determinant of antibody specificity (a single FdFragment may be associated with up to ten different light chains withoutaltering antibody specificity) and Fd fragments retain epitope-bindingability in isolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(Frs), which maintain the tertiary structure of the paratope (see, ingeneral, Clar, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of nonspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or Fr and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornonhuman sequences. The present invention also includes so-called singlechain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to cellular adhesion molecules. These polypeptides maybe derived also from sources other than antibody technology. Forexample, such polypeptide binding agents can be provided by degeneratepeptide libraries which can be readily prepared in solution, inimmobilized form or as phage display libraries. Combinatorial librariesalso can be synthesized of peptides containing one or more amino acids.Libraries further can be synthesized of peptoids and non-peptidesynthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe cellular adhesion molecule. This process can be repeated throughseveral cycles of reselection of phage that bind to the cellularadhesion molecule. Repeated rounds lead to enrichment of phage bearingparticular sequences. DNA sequences analysis can be conducted toidentify the sequences of the expressed polypeptides. The minimal linearportion of the sequence that binds to the cellular adhesion molecule canbe determined. One can repeat the procedure using a biased librarycontaining inserts containing part of all of the minimal linear portionplus one or more additional degenerate residues upstream or downstreamthereof. Yeast two-hybrid screening methods also may be used to identifypolypeptides that bind to the cellular adhesion molecules. Thus,cellular adhesion molecules, or a fragment thereof, can be used toscreen peptide libraries, including phage display libraries, to identifyand select peptide binding partners of the cellular adhesion molecules.

In practicing the methods of the present invention, it is required toobtain a level of sCD40L in an individual. Soluble CD40L is well-knownto those of ordinary skill in the art. The level of sCD40L for theindividual can be obtained by any art recognized method. Typically, thelevel is determined by measuring the level of the marker in a bodyfluid, for example, blood, lymph, saliva, urine and the like. The levelcan be determined by ELISA, or immunoassays or other conventionaltechniques for determining the presence of the marker. Conventionalmethods include sending samples of a patient's body fluid to acommercial laboratory for measurement.

The invention also involves comparing the level of sCD40L for theindividual with a predetermined value. The predetermined value can takea variety of forms. It can be single cut-off value, such as a median ormean. It can be established based upon comparative groups, such as wherethe risk in one defined group is double the risk in another definedgroup. It can be a range, for example, where the tested population isdivided equally (or unequally) into groups, such as a low-risk group, amedium-risk group and a high-risk group, or into quadrants, the lowestquadrant being individuals with the lowest risk and the highest quadrantbeing individuals with the highest risk.

The predetermined value can depend upon the particular populationselected. For example, an apparently healthy, nonsmoker population withno detectable disease and no prior history of a cardiovascular disorderwill have a different ‘normal’ range of sCD40L than will a smokingpopulation or a population the members of which have had a priorcardiovascular disorder. Accordingly, the predetermined values selectedmay take into account the category in which an individual falls.Appropriate ranges and categories can be selected with no more thanroutine experimentation by those of ordinary skill in the art.

The preferred body fluid is blood. For sCD40L, one important cut-off fora population of apparently healthy, nonsmokers is 2.9 ng/mL or below.Another important cut-off for sCD40L is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10.0, 10.5 or 11.0 ng/mL. In characterizing risk,numerous predetermined values can be established.

There presently are commercial sources which produce reagents for assaysfor sCD40L. These include, but are not limited to, BenderMedSystems(Vienna, Austria), and Abbott Pharmaceuticals (Abbott Park, Ill.).

In preferred embodiments the invention provides novel kits or assayswhich are specific for, and have appropriate sensitivity with respectto, predetermined values selected on the basis of the present invention.The preferred kits, therefore, would differ from those presentlycommercially available, by including, for example, different cut-offs,different sensitivities at particular cut-offs as well as instructionsor other printed material for characterizing risk based upon the outcomeof the assay.

As discussed above the invention provides methods for evaluating thelikelihood that an individual will benefit from treatment with an agentfor reducing risk of a future cardiovascular disorder. This method hasimportant implications for patient treatment and also for clinicaldevelopment of new therapeutics. Physicians select therapeutic regimensfor patient treatment based upon the expected net benefit to thepatient. The net benefit is derived from the risk to benefit ratio. Thepresent invention permits selection of individuals who are more likelyto benefit by intervention, thereby aiding the physician in selecting atherapeutic regimen. This might include using drugs with a higher riskprofile where the likelihood of expected benefit has increased.Likewise, clinical investigators desire to select for clinical trials apopulation with a high likelihood of obtaining a net benefit. Thepresent invention can help clinical investigators select suchindividuals. It is expected that clinical investigators now will use thepresent invention for determining entry criteria for clinical trials.

In another surprising aspect of the invention, it has been discoveredthat sCD40L has predictive value independent of other known predictorsof future adverse cardiovascular disorders. Thus, the present inventiondoes not involve simply duplicating a measurement that previously couldbe made using other predictors. Instead, levels of sCD40L are additiveto prior art predictors. Prior art predictors include markers ofsystemic inflammation, such as C-Reactive Protein, cytokines, andcellular adhesion molecules. Cytokines are well-known to those ofordinary skill in the art and include human interleukins 1-17. Cellularadhesion molecules are well-known to those of ordinary skill in the artand include integrins, ICAM-1, ICAM-3, BL-CAM, LFA-2, VCAM-1, NCAM, andPECAM. Prior art predictors also include cholesterol.

In a further surprising aspect of the invention, it has been discoveredthat sCD40L has predictive value on the presence of vascularintra-plaque lipid accumulation in individuals at an elevated risk ofdeveloping a cardiovascular disorder. As used herein, individuals “atrisk of developing a cardiovascular disorder” are a category of subjectsdetermined according to conventional medical practice. (See, e.g.,Harrison's Principles of Experimental Medicine, 15th Edition,McGraw-Hill, Inc., New York).

Typically, an individual at risk of developing a cardiovascular disorderhas one or more risk factors associated with cardiovascular disease.Such risk factors include family history of a cardiovascular disorder,hypertension, hypercholesterolemia, diabetes, smoking, atherosclerosis,etc. In addition, atrial fibrillation, or recent stroke and/ormyocardial infarction are important risk factors. Previously,determination of vascular intra-plaque lipid accumulation in individualsat risk of developing a cardiovascular disorder could only beaccomplished using expensive, and in certain cases limiting, technology(e.g., MRI).

It is known in the art that a major factor invoking coronary thrombosisis disruption of an atherosclerotic plaque. As explained elsewhereherein, determining intra-plaque lipid accumulation in individuals atrisk of developing a cardiovascular disorder is an important step indetermining plaque vulnerability (i.e. plaques at risk of disruption),and thus assessing risk for the future occurrence of a thrombotic event.Studies comparing intact and disrupted plaques have been used to definethe characteristics of vulnerable plaques. The characteristics are alipid core occupying over 50% of overall plaque volume, a thin plaquecap, a large absolute number and density of macrophages, and a reductionin the smooth muscle content of the plaque. Thus, after determiningintra-plaque lipid accumulation in individuals at risk of developing acardiovascular disorder according to the methods of the presentinvention, one of skill in the art could evaluate whether the plaque isvulnerable and devise appropriate theraputic/interventional regimens toprevent the occurrence of a subsequent thrombotic event in theindividual.

The invention also involves a method for treating subjects, withtherapies, to prevent cardiovascular disorders. An agent selected fromthe group consisting of an anti-inflammatory agent, an antithromboticagent, an anti-platelet agent, a fibrinolytic agent, a lipid reducingagent, a direct thrombin inhibitor, a glycoprotein IIb/IIIa receptorinhibitor, or an agent that binds to cellular adhesion molecules andinhibits the ability of white blood cells to attach to such molecules,and/or any combinations thereof, is administered to a subject who has anabove-normal level of a marker of systemic inflammation. The agent isadministered in an amount effective to lower the risk of the subjectdeveloping a future cardiovascular disorder. In some embodiments theagent is a non-aspirin anti-inflammatory agent. Agents are describedelsewhere herein.

An effective amount is a dosage of the agent sufficient to provide amedically desirable result e.g., reduction in risk. The effective amountwill vary with the particular condition being treated, the age andphysical condition of the subject being treated, the severity of thecondition, the duration of the treatment, the nature of the concurrenttherapy (if any), the specific route of administration and the likefactors within the knowledge and expertise of the health practitioner.For example, an effective amount can depend upon the degree to which anindividual has abnormally elevated levels of sCD40L. It should beunderstood that the agents used according to the invention are intendedto lower the risk of a cardiovascular disorder, that is, they are usedprophylactically. Thus, an effective amount is that amount which canlower the risk of, slow or perhaps prevent altogether the development ofa cardiovascular disorder.

Generally, doses of active compounds would be from about 0.01 mg/kg perday to 1000 mg/kg per day. It is expected that doses ranging from 50-500mg/kg will be suitable, preferably orally and in one or severaladministrations per day. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of compounds.

When administered, the pharmaceutical preparations of the invention areapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptably compositions. Such preparations mayroutinely contain salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents. When used inmedicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically-acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

The anti-inflammatory agents may be combined, optionally, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the anti-inflammatory agent,which is preferably isotonic with the blood of the recipient. Thisaqueous preparation may be formulated according to known methods usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation also may be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Carrier formulation suitable fororal, subcutaneous, intravenous, intramuscular, etc. administrations canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular drug selected, theseverity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, intradermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Intravenous orintramuscular routes are not particularly suitable for long-term therapyand prophylaxis. They could, however, be preferred in emergencysituations. Oral administration will be preferred for prophylactictreatment because of the convenience to the patient as well as thedosing schedule.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing theanti-inflammatory agent into association with a carrier whichconstitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing theanti-inflammatory agent into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the anti-inflammatory agent. Other compositionsinclude suspensions in aqueous liquids or non-aqueous liquids such as asyrup, elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of an agent of the present invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are: lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono- di- and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189,and 5,736,152, and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-basedhardware delivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

The invention will be more fully understood by reference to thefollowing example. This example, however, is merely intended toillustrate the embodiments of the invention and is not to be construedto limit the scope of the invention.

EXAMPLES Example 1 High Plasma Concentrations of sCD40L are Associatedwith Increased Vascular Risk in Apparently Healthy Women

We performed a prospective, nested case control analysis amongparticipants in the Women's Health Study (WHS), an ongoing primaryprevention trial evaluating the efficiency of vitamin E and low doseaspirin in 28,263 middle aged American women with no history ofcardiovascular disease or cancer.¹¹ Blood samples were collected in EDTAand stored in liquid nitrogen until analysis. For this study, 130 womenwho subsequently developed either non-fatal myocardial infarction orstroke, or died from acute cardiovascular events during the initialfour-year follow-up period were selected as ‘case’ subjects. A committeeof physicians using standardized procedures classified endpoints. Foreach confirmed case, a ‘control’ participant of same age (±2 years),similar smoking status (former, current, never) and who remained free ofreported cardiovascular disease was selected.

Measurement of baseline plasma sCD40L concentrations used an ELISA(BenderMedSystems; Vienna, Austria). Briefly, diluted (1:5) plasmasamples were applied in triplicate to 96-well plates precoated withmouse-anti-human CD40L antibody and mixed (1:2) with ahorseradish-peroxidase-labeled secondary mouse-anti-human CD40L antibody(2 h). Subsequently, plates were washed and antibody binding determinedby colorimetry employing TMB substrate. Absorbance was read at 650 nmand plasma concentrations of sCD40L determined by comparison with serialdilutions of recombinant human CD40L. The analysis was performed in ablinded fashion. The intra-assay variation among the triplicates for allsamples was less than 15 percent. Lipid levels were measured in alaboratory which participates in the Centers for Disease ControlStandardization.

Means and proportions for baseline clinical characteristics of the studyparticipants were computed and compared using either Student's T-test orthe chi-square statistic. Relative risk of developing futurecardiovascular events associated with increasing levels of sCD40L atbaseline were then computed in a series of logistic regression analysiswhich divided the study sample according to the 50^(th), 75^(th),90^(th,) 95^(th), and 99^(th) percentile cutpoints of the controldistribution for sCD40L. All p values are two-tailed and all confidenceintervals computed at the 95% level.

Results

Table I shows the baseline clinical characteristics of the studyparticipants. As expected, women who developed cardiovascular diseaseduring follow-up were more likely at study entry to be obese,hypertensive, diabetic, or have a family history of prematureatherosclerosis compared to women who remained free of disease. LDLcholesterol and triglyceride levels were higher at baseline among cases,whereas HDL cholesterol levels were lower (all p≦0.01). Use of hormonereplacement therapy did not differ significantly between the two groups.

Overall, plasma levels of sCD40L at baseline among cases exceeded thatin controls (2.86±0.35 vs. 2.09±0.19 ng/mL; p≦0.02). This differenceresulted almost completely from an excess of particularly high valuesamong the case subjects. The great majority of cases and controlsubjects had similar levels of sCD40L at study entry (FIG. 1). However,11 cases had baseline levels of sCD40L in excess of the 99^(th)percentile cutpoint for the control distribution as compared to only 1study subject in the control group (p≧0.01).

Relative risks of developing future cardiovascular events, according tothe pre-specified cutpoints defined by the distribution of the studycontrols, rose with increasing concentrations of sCD40L and becamestatistically significant with levels of sCD40L in excess of the 95^(th)and 99^(th) percentile cutpoints (RR: 3.29 (p≦0.02) and 11.83 (p≦0.01),respectively) (Table II).

An additional post-hoc analysis was performed comparing clinicalcharacteristics among the 12 participants with levels of sCD40L inexcess of the 99^(th) percentile cutpoint to the 248 participants withlower levels. Age, smoking, body mass index, LDL, and HDL cholesterollevels and hormone replacement therapy were similar between these twostudy groups (Table III). Study participants with particularly elevatedlevels of sCD40L had somewhat higher rates of hypertension and a familyhistory of premature coronary artery disease, but neither of thesedifferences achieved statistical significance. None of the 12 women withmarkedly elevated baseline levels of sCD40L had diabetes. Moreover,there were no significant differences in the time from randomization tothe time of the cardiovascular event between the 11 cases with extremesCD40L levels and the remaining cases with lower sCD40L (15.9 vs. 19.5months, p=0.3). Assignment to aspirin vs. placebo presumably did notaffect our observation, since within the group of 12 subjects showingthe highest sCD40L concentrations six were randomly assigned to aspirinand six to placebo. In addition, the bloods assayed for sCD40L weredrawn before randomization.

Previous reports on this cohort have documented association betweenplasma levels of C-reactive protein, IL-6, serum amyloid A and ICAM-1with increased cardiovascular risk.¹² However, we observed nosignificant correlation between these parameters and sCD40L.

Discussion

In this prospective, nested case control study of apparently healthymiddle aged women, markedly elevated plasma concentrations of sCD40L atbaseline (≧5.5 ng/mL) foretold a significantly increased risk of futurecardiovascular events. Previous studies demonstrated that patients withunstable angina had significantly raised serum levels of sCD40L whencompared with patients with stable angina and controls.¹⁰ In thiscircumstance activated platelets and/or T lymphocytes may release sCD40Lsecondarily. The present study, however, demonstrates elevation ofsCD40L concentrations in some women before events that may result fromacute thrombosis.

Little is known regarding the mechanisms yielding release of solubleforms of CD40L. Potential source(s) for sCD40L in plasma includeplatelets and T lymphocytes as well as mononuclear phagocytes andendothelial cells.¹⁻⁵ The tendency of family history for cardiovasculardisease to correlate with enhanced sCD40L plasma levels suggests thatgenetic factors, might contribute to our observation. We found noassociation between sCD40L and C-reactive protein, IL-6, and ICAM-1levels. It is therefore implied that CD40/CD40L-independent mechanismsmay well pertain to women developing cardiovascular complicationsdespite low sCD40L levels. Our discoveries suggest that high plasmaconcentrations of sCD40L reflect aspects of risk distinct from thosegauged by other inflammatory markers.

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12. Ridker P M, Hennekens C H, Buring J E, et al. N Engl J Med. 2000;342:836-43.

DETAILED DESCRIPTION OF THE DRAWING

FIG. 1. Baseline serum concentrations of sCD40L among middle agedhealthy women which either stayed free of (controls, n=130) or developedcardiovascular events (cases, n=130). Samples were analyzed intriplicates, mean values are shown. The dotted line depicts the 99^(th)percentile cutpoint for the control distribution. TABLE I Baselinecharacteristics of study participants. Controls Cases (N = 130) (N =130) P-value Age, years 60.3 60.3 Matching criteria Smoking Status (%)Matching criteria Current 26.9 26.9 Former 31.6 31.6 Never 41.5 41.5Body Mass Index (kg/m²) 25.7 27.6 0.004 Hypertension (%) 34.9 56.9 0.001Family history of CAD (%)* 10.8 22.7 0.01 Diabetes (%) 3.1 10.8 0.02Current HRT** (%) 40.0 44.6 0.1 LDL (mg/dL) 118.4 128.5 0.02 HDL (mg/dL)48.4 42.6 0.01 Triglycerides (mg/dL) 136.5 161.0 0.01 sCD40L (ng/mL)2.09 2.86 0.02*Before age 60;**HRT, hormone replacement therapy

TABLE II Baseline characteristics of study participants. Cutpoint sCD40LControls Cases P- (percentile) (ng/mL) % (N) % (N) RR 95% CI value50^(th) >1.76 50.4 (65) 50.8 (66) 1.02 0.62-1.65 0.95 75^(th) >2.15 24.9(32) 30.8 (40) 1.35 0.78-2.33 0.29 90^(th) >2.92 10.1 (13) 17.7 (23)1.92 0.93-3.98 0.08 95^(th) >3.71 5.0 (6) 13.9 (18) 3.29 1.26-6.59 0.0299^(th) >5.54 0.8 (1)  8.5 (11) 11.83 1.50-93.0 0.01

TABLE III Clinical characteristics of study participants with sCD40Lconcentrations above 99^(th) percentile of the control distribution(≧5.54 ng/mL sCD40L) sCD40L, >99^(th) sCD40L, ≦99^(th) (N = 12) (N =248) P-value Age, years 63.1 60.2 0.3 Smoking Status (%) 0.7 Current25.0 27.1 Former 33.3 47.7 Never 41.7 31.2 Body Mass Index (kg/m²) 27.326.7 0.7 Hypertension (%) 58.3 45.1 0.4 Family history of CAD (%)* 30.016.0 0.2 Diabetes (%) 0 7.3 0.9 Current HRT** (%) 50.0 41.7 0.8 LDL(mg/dL) 126.9 127.7 0.9 HDL (mg/dL) 49.8 48.4 0.7 Triglycerides (mg/dL)165.5 174.3 0.8*Before age 60;**HRT, hormone replacement therapy

Example 2 HMG-CoA Reductase Inhibitors Limit CD40 and CD40L Expressionin Human Vascular Cells

This study tested the hypothesis that HMG-CoA reductase inhibitors(statins) can diminish the expression of the receptor/ligand dyad onthese cells in vitro as well as of sCD40L plasma levels in vivo, andthat oxidatively modified LDL induces the expression of CD40/CD40L oncells involved in atherosclerosis, namely human vascular EC and SMC, aswell as MØ.

Materials and Methods

Materials

Native and oxidized (5 μM CuSO₄, 37° C., 24 h) LDL (TBARS: 0.4 and 8.3nM MDA/mg protein, respectively) were obtained from BiomedicalTechnologies, Inc. (Stoughton, Mass.). Human recombinant IL-1β, TNFα,and IFNγ were obtained from Endogen (Woburn, Mass.).

Cell Isolation and Culture

Human vascular EC and SMC were isolated from saphenous veins andcultured as described previously.^(4,26) Mononuclear phagocytes (MØO)were isolated from leukocyte concentrates by density gradientcentrifugation employing Lymphocyte Separation Medium (Organon-Teknika,Durham, N.C.) and were cultured (10 days) in RPMI 1640 (BioWhittaker,Walkersville, Md.) containing 2% human serum (Sigma; St. Louis, Mo.).²⁶All cell types were cultured 24 h before and during the experiment inmedia lacking serum. Viability of the cultures was determined by trypanblue (Sigma, St. Louis, Mo.) exclusion count as well as anoligonucleosome formation assay (Cell Death Detection ELISA, BoehringerMannheim, Germany).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA isolated from cultured EC, SMC, or MØ employing RNAzol(Tel-Test; Friendswood, Tex.) was assessed for purity and yieldspectrophotometrically (2100 Bioanalyzer, Agilent Technologies,Wilmington, Del) and was reverse-transcribed (2 μg total RNA; 50min, 42°C.) employing Superscript II Reverse Transcriptase (LifeTechnologies,Carlsbad, Calif.). PCR was performed for 35 cycles at 95° C. (120 sec),62° C. (120 sec), and 72° C. (180 sec, 2 sec prolongation per cycle)after hot start, employing primers for CD40 or CD40L previouslydescribed.⁴ Semiquantitative PCR studies employing 20, 25, 30, 35, and40 cycles verified that the conditions used yielded PCR products withinthe exponential range of amplification and were optimized forsignal:background ratios . PCR products were analyzed on ethidiumbromide-containing 1.3% agarose gels and visualized by UVtransillumination. Loading of equal template amounts was verified byRT-PCR for GAPDH. Mock RT reactions, either lacking reversetranscriptase or employing H₂O as template, demonstrated specificity ofthe signals obtained.

Western Blot

Culture lysates (50 μg total protein/lane) and supernatants wereseparated by SDS-PAGE and blotted to polyvinylidene difluoride membranes(Bio-Rad, Hercules, Calif.) using a semi-dry blotting apparatus (3mA/cm², 30 min; Bio-Rad, Hercules, Calif.). Blots were blocked andprimary (mouse-anti-human CD40 or CD40L; both 1:1,000; PharMingen, SanDiego, Calif.) antibodies were added in 5% defatted dry milk/PBS/0.1%Tween 20. After 1 h, blots were washed three times (PBS/0.1% Tween 20)and secondary, peroxidase-conjugated, goat-anti-mouse antibody (JacksonImmunoresearch, West Grove, Pa.) was added (1 h). Finally, blots werewashed and immunoreactive proteins were visualized using the Westernblot chemiluminescence system (NEN™, Boston, Mass.). Data were verifiedby employing anti-CD40/-CD40L antibodies from Santa Cruz (Santa Cruz,Calif.).

Flow Cytometry

Human vascular EC, SMC, or MØ were washed with ice-cold PBS, harvestedby trypsinization, and fixed (PBS/4% paraformaldehyde, 15min).Subsequently, the cells were washed once with PBS/2% BSA before beingincubated (1 h, 4° C.) with either buffer alone or FITC-conjugatedcontrol IgG, mouse-anti-human CD40, or mouse-anti-human CD40L antibody(1 μg/ml; PharMingen, San Diego, Calif.). Finally, cells were washedwith PBS/2% BSA and analyzed in a Becton Dickinson FACSCAN® flowcytometer employing CELLQUEST® software (Becton Dickinson; San Jose,Calif.). At least 20,000 viable cells per condition were analyzed.

Patient Studies

Whole blood (10 mL) was collected in EDTA from 27 patients presentingfor coronary arteriography who had at least a 30% stenosis in onecoronary artery. The cohort was divided into patients who were or werenot treated with any statin at the time of catheterization. Blood wasdrawn at baseline (0 month) and final follow-up visit (6 months), andplasma was stored at −70° C. All subjects were studied in the fastingstate. Written informed consent was obtained from all subjects and thestudy was approved by the Human Research Committee of Brigham andWomen's Hospital. Plasma lipids as well as IL-1β, IL-6, TNFα, IFNγ,sVCAM, C-reactive protein (CRP), and sCD40L were measured by ELISA(Sigma, St. Louis, Mo.; Endogen, Woburn, Mass.; BenderMedSystems,Vienna, Austria). The two groups did not differ significantly in age,gender, diabetes mellitus, smoking, triglycerides, or HDL (Table I). Thestatin-treated group had significantly lower total cholesterol and LDL,as expected.

Ex vivo Fibrin Clot Formation

Blood was collected from mice by retro-orbital bleeding into 0.1 volumeof 0.13M trisodium citrate using non-coated capillary tubes.Platelet-rich plasma (PRP) was prepared by centrifugation (500×g, 5 min,20° C.) and fibrin clot formation was examined using a modification of amicrotiter-plate clot lysis assay described previously.²⁷ Clots wereprepared with 2.94 μM fibrinogen, 0.24 μM plasminogen, 36 μM t-PA, 3.8nM thrombin, and 5.3 mM CaCl₂ (all final concentrations). PRP (finalconcentration of 10% (v/v)) was incorporated into clots. Clot formationwas monitored at 405nm for up to 15 min.

Statistical Analysis

Data are presented as mean+SD and groups were compared using theStudent's t-test. A value of p≦0.05 was considered significant.

Results

HMG-CoA Reductase Inhibitors Diminish the Expression of CD40 and CD40Lon Human Vascular Endothelial and Smooth Muscle Cells, as Well asMacrophages in vitro

Exposure to HMG-CoA reductase inhibitors concentration-dependentlydiminished the expression of both CD40 and CD40L in human vascular EC,SMC, and MØ. At concentrations ≧2 nM cerivastatin diminished theconstitutive as well as IL-1β/TNFα/IFNγ-induced expression of thereceptor in EC. Maximal inhibition was achieved at 10-50 nMcerivastatin. Mevalonate reversed the diminished expression of CD40 andCD40L by statins. Parallel studies employing oxLDL as a stimulus or SMCor MØ as the cell type yielded similar results. To determine whether themodulation of the CD40/CD40L expression extends to other statins,parallel experiments employed atorvastatin or simvastatin. These HMG-CoAreductase inhibitors similarly reduced CD40/CD40L expression, butrequired higher concentrations (100 nM). Re-development of the Westernblots with a mouse-anti-human GAPDH antibody, providing similarband-intensities across the blots, verified equal loading among thelanes, and also suggested that statin treatment per se did not affectWestern blot analysis. Of note, neither statin affected cell number orviability at the concentrations analyzed (up to 250 nM), as determinedby trypan blue exclusion cell count, as well as mono/oligonucleosomeformation.

HMG-CoA reductase inhibitors moreover diminished CD40 and CD40L mRNAexpression in human vascular EC and MO stimulated either withpro-inflammatory cytokines (IL-1β/TNFα/IFNγ) or oxLDL. Atorvastatin orsimvastatin yielded similar results. Mevalonate reversed the diminishedexpression of CD40 and CD40L transcripts. Parallel studies analyzing theexpression of GAPDH transcripts demonstrated application of equalamounts of reverse transcribed mRNA to each reaction and furthermoresuggested that treatment with statins per se did not affect the RT-PCR.

In accord with the findings for whole cell lysates, cerivastatinconcentration-dependently diminished the cell surface expression of bothCD40 and CD40L on human vascular EC, as well as MØ. Notably, thecerivastatin concentrations required for minimal and maximal reductionin CD40 and CD40L surface expression resembled those observed for wholecell lysates in the Western blot as well as the RT-PCR studies. Allthree statins acted similarly, but required different concentrations toachieve equal reduction in CD40/CD40L expression(cerivastatin>simvastatin=atorvastatin).

Since CD40L can also be shed as a biologically active soluble form,sCD40L, we tested whether treatment of EC, SMC, and MØ with HMG-CoAreductase inhibitors affected the secretion of sCD40L. Indeed,cerivastatin treatment markedly lowered sCD40L concentrations inculture.

Combined, RT-PCR, Western blot, and FACS studies suggest that oxLDLinduces and statins diminish the cell surface expression and/or releaseof CD40 or CD40L by regulating gene activity rather than intracellulartranslocation.

HMG-CoA Reductase Inhibitors Diminish sCD40L Plasma Levels in Humans

To assess the potential clinical relevance of these in vitro findings,we performed a pilot study to determine whether treatment of patientswith HMG-CoA reductase inhibitors diminished sCD40L plasma levels.Plasma of statin-treated atherosclerotic subjects had significantlylower levels of sCD40L compared to non-treated patients (8.3±3.1 ng/ml(n=11) vs. 13.1±2.5 ng/ml (n=16); p<0.05). The individual sCD40L levelsdid not vary significantly if the treatment status of the patient atbaseline and 6 month follow-up did not change. However, sCD40L plasmalevels at 6 month follow-up decreased markedly in patients changed tostatin treatment (13.1±5.74 ng/ml vs. 5.21±2.36 ng/ml (n=4); p<0.05).Plasma levels of IL-1β, IL-6, TNFα, IFNγ, and sVCAM-1 did not changewith statin treatment. Plasma concentrations of C-reactive protein (CRP)were lower in the statin-treated group, although the difference did notachieve statistical significance.

Oxidized LDL Induces Expression of CD40 and CD40L in Human VascularCells

Oxidatively modified LDL (oxLDL) concentration-dependently enhanced thefaint constitutive expression of CD40 and CD40L protein in humanvascular EC and MØ. Augmentation of basal CD40/CD40L expression oneither cell type required 1-3 μg oxLDL/ml. Maximal expression of CD40(5.1±1.1 fold above non-stimulated control; n=4) and CD40L (4.2±2.2fold; n=3) immunoreactive protein was achieved with 10-30 μg oxLDL/ml.Native LDL also induced expression of the receptor (2.8±1.1 fold; n=2)and ligand (2.2±0.8 fold; n=2), although to a lesser extent.Furthermore, oxLDL concentration-dependently augmented the expression ofCD40 and CD40L mRNA in human vascular EC or MØ. Parallel studiesanalyzing the expression of GAPDH transcripts demonstrated applicationof equal amounts of reverse transcribed mRNA amounts to each reactionand furthermore suggested that oxLDL stimulation per se did not affectthe RT-PCR.

Deficiency of CD40L Prolongs Time of Coagulation of Murine Blood ex vivo

In light of recent data implicating CD40L in thrombosis and thestatin-mediated lowering of plasma sCD40L levels shown above, we furthertested the hypothesis that diminished expression of CD40L affects bloodcoagulation. Indeed, platelet-rich plasma from CD40L-deficient miceshowed delayed coagulation in a fibrin clot formation assay whencompared to wildtype mice preparations, supporting a role for CD40L inblood coagulation. These observations on the role of CD40L in bloodcoagulability provide a pathway by which reduction in CD40 signaling viaHMG-CoA reductase inhibitors might reduce thrombotic complications ofatherosclerosis.

Discussion

Clinical benefits in patients with average or below average LDL levelsand reduced cardiovascular risk independent of the degree ofLDL-lowering in a consistent series of previous clinical trials havehighlighted the potential clinical relevance of the putative“pleiotropic” effects of statins.^(21-24,28) In addition to theirlipid-lowering effects, numerous clinical and experimental studies havesuggested anti-inflammatory pathways of statins, such as diminishedexpression of chemokines, major histocompatibility complex II molecules,matrix-degrading enzymes, and the procoagulant tissue factor, as well asthe augmented expression of nitric oxide.^(22,23,25,28) Moreover,treatment of Watanabe heritable hyperlipidemic rabbits with HMG-CoAreductase inhibitors diminishes the expression of numerouspro-atherogenic inflammatory mediators in vivo.^(29,30) Although statinslowered lipids only modestly in these rabbits lacking LDL-receptors,these in vivo observations could not conclusively distinguish the degreeto which effects on lipoproteins account for the anti-inflammatoryeffects observed. The present report provides evidence for a novelanti-inflammatory pathway by which statins may act both dependently andindependently of lipid-lowering. Three members of this drug class(cerivastatin, atorvastatin, or simvastatin) significantly diminishedthe constitutive as well as cytokine-induced expression of CD40 andCD40L protein and transcript in cell types implicated inatherosclerosis, namely human vascular EC, SMC, and MØ, arguing for alipid-lowering independent function of statins. However, theidentification of oxLDL as an inducer of CD40 and CD40L in these celltypes further suggests that statins might affect CD40/CD40L expression,at least in part, also via their lipid-lowering properties. Of note,previous studies have colocalized oxLDL with CD40 and CD40L within earlyhuman atherosclerotic lesions,³¹ a finding in accord with our hypothesisthat oxLDL provides an initial signal for the expression of the CD40receptor/ligand dyad in atherosclerotic plaques.

The pilot observation that patients treated with statins have diminishedlevels of sCD40L supports the clinical relevance of the present in vitroobservations. Several cell types might give rise to sCD40L. Plateletsrelease sCD40L upon ligation of the thrombin receptor in vitro as wellas upon thrombus formation in vivo.^(32,33) However, as suggested by ourown and other previous studies, other cell types, including EC, MØ, andT lymphocytes, might also generate sCD40L.^(11,12,17,18)

The present observation that CD40L-deficient platelet-rich plasma clotsmore slowly than preparations from wild type mice suggests that CD40L,in its membrane-bound and/or soluble form, modulates thrombosis, acrucial determinant of cardiovascular risk. The finding that CD40L canactivate platelets by functioning as an α_(IIb)β₃ ligand furthersupports this hypothesis.³⁵

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5. Gaweco A S, Wiesner R H, Yong S, et al. Liver Transpl. Surg. 1999;5:1-7.

6. Afford S C, Randhawa S, Eliopoulos A G, et al. J Exp Med. 1999;189:441-446.

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9. Miller D L, Yaron R, Yellin M J. J Leukoc Biol. 1998; 63:373-379.

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11. Schönbeck U, Libby P. Cell Mol Life Sci. 2001; 58:4-43.

12. Schönbeck U, Libby P. Circ Res. 2001; 89:1092-1103.

13. Mach F, Schönbeck U, Sukhova G K, et al. Nature. 1998; 394:200-203.

14. Lutgens E, Gorelik L, Daemen M J, et al. Nat Med. 1999; 5:1313-1316.

15. Schönbeck U, Sukhova G K, Shimizu K, et al. Proc Natl Acad Sci U SA. 2000; 97:7458-7463.

16. Lutgens E, Cleutjens K B, Heeneman S, et al. Proc Natl Acad Sci U SA. 2000; 97:7464-7469.

17. Graf D, Muller S, Korthauer U, et al. Eur J Immunol. 1995;25:1749-1754.

18. Ludewig B, Henn V, Schroder J M, et al. Eur J Immunol. 1996;26:3137-3143.

19. Aukrust P, Muller F, Ueland T, et al. Circulation. 1999;100:614-620.

20. Schönbeck U, Varo N, Libby P, et al. Circulation. 2001;104:2266-2268.

21. Vaughan C J, Murphy M B, Buckley B M. Lancet. 1996; 348:1079-1082.

22. Dangas G, Smith D A, Unger A H, et al. Thromb Haemost. 2000;83:688-692.

23. Ni W, Egashira K, Kataoka C, et al. Circ Res. 2001; 89:415-421.

24. Heeschen C, Hamm C W, Laufs U, et al. Circulation. 2002;105:1446-1452.

25. Kwak B, Mulhaupt F, Myit S, et al. Nat Med. 2000; 6:1399-1402.

26. Schönbeck U, Mach F, Sukhova G K, et al. J Exp Med. 1999;189:843-853.

27. Robbie L A, Booth N A, Croll A M, et al. Thromb Haemost. 1993;70:301-306.

28. Libby P, Aikawa M, Schönbeck U. Biochim Biophys Acta. 2000;1529:299-309.

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30. Aikawa M, Rabkin E, Okada Y, et al. Circulation. 1998; 97:2433-2444.

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35. Andre P, Prasad K S, Denis C V, et al. Nat Med. 2002; 8:247-252.

Example 3 Soluble CD40 Ligand Levels Indicate Lipid Accumulation inCarotid Atheroma

A major factor invoking coronary thrombosis is disruption of anatherosclerotic plaque. Studies comparing intact and disrupted plaqueshave been used to define the characteristics of vulnerable plaques i.e.those at risk of disruption. The characteristics are a lipid coreoccupying over 50% of overall plaque volume, a thin plaque cap, a largeabsolute number and density of macrophages, and a reduction in thesmooth muscle content of the plaque. Such vulnerable plaques make up asmall proportion of all the plaques present in most individuals.Angiographic stenosis, however, does not predict vulnerability becausethere is no relation between core size or plaque size with stenosis. Alarge proportion of disruption episodes go unnoticed clinically becausethe thrombus does not sufficiently encroach on the lumen to causeischaemia. These subclinical episodes, however, will invoke plaquegrowth. Plaque disruption is followed by a smooth muscle proliferativerepair response analogous to that occurring after angioplasty. In bothsituations, exuberant repair leads to post event stenosis.Reconstruction of coronary lesions at autopsy shows that 70% of highgrade stenosis (angiographic >50% diameter) have had an episode ofhealed disruption. Such data highlight the role of plaque disruption inthe generation of advanced stenotic lesions irrespective of whether anacute clinical event occurred.

In summary, the structure and the dynamic biology of the atheroma,rather than the severity of stenosis, largely determine cardiovascularevents. Large lipid pools and thin fibrous caps characterize vulnerableplaque, and inflammatory mechanisms play a pivotal role in determiningplaque stability.^(1,2) Much of our knowledge of the unstable atheromaderives from post-mortem examination, and less is known about therelationships of inflammatory mechanisms and lesion structure in vivo.

Evidence from animal studies supports the importance of CD40 ligand asinhibition of CD40 signaling in atherosclerosis-prone mice reduced thesize and lipid content of aortic lesions, and yielded a relativeincrease in smooth muscle content and fibrillar collagen.⁹ Moreover, asdiscussed elsewhere herein (see Example 1), elevated plasma levels ofsoluble CD40 ligand at baseline predict prospectively cardiovascularevents among apparently healthy women.

Recent advances in magnetic resonance imaging (MRI) have permittednon-invasive assessment of carotid plaque composition.¹¹⁻¹³Specifically, use of a custom-made phased array carotid coil hasdemonstrated high levels of agreement between carotid MRI findings andresults at histology among 22 patients undergoing carotid endarterectomy(89% agreement; kappa=0.83; 95% confidence interval 0.67-1.0).¹⁴Furthermore, high-resolution carotid MRI accurately detects intra-plaquelipid-rich cores.^(12,13,15,16)

This study tested the hypothesis that elevated plasma levels of solubleCD40 ligand correlated with features suggestive of lipid-rich cores onhigh-resolution carotid MRI.

Methods

We invited men and women with stenoses greater than or equal to 30percent in either internal or common carotid artery by carotidultrasonography to participate in the study, from January 2001 toJanuary 2002. Any patient with a pacemaker or implantable cardioverterdefibrillator was excluded, as well as patients who had receivedsurgical clips or coronary stents in the previous two months. Patientsrequiring systemic corticosteroids for a systemic inflammatory conditionwere also excluded. The study population comprised the 49 consecutivepatients who gave informed written consent to participate in the study.The study was approved by the Human Research Committee of Brigham andWomen's Hospital.

A detailed medical history including prior cardiovascular history, riskfactors, and medication use was recorded by a study physician for eachparticipant. A blood sample was drawn by non-traumatic venipuncture,centrifuged, and the plasma stored in EDTA at −80° C.

MRI protocol: The patients underwent high resolution MRI of the carotidarteries using a dedicated phased array carotid coil (IGC, Inc.) on a1.5T Signa CV/i MRI scanner (GE Medical Systems, Milwaukee, Wis.). 3Dtime-of-flight images, moderately proton density weighted images, andfat-suppressed moderately T2-weighted images were obtained. For the 3Dtime-of-flight sequences, parameters were as follows: echo time (TE) 3.5ms, repetition time (TR) 33-40 ms, flip angle 25 degrees, bandwidth15.63 kHz, field of view 12-14 cm, slice thickness 2 mm interpolated to1 mm, 32 slices, acquired matrix 256×256, reconstructed matrix 512×512,with one excitation. For the moderately proton density weighted sequencea TE of 21-22 ms was used, and for the moderately T2-weightedacquisition a TE of 53-58 ms was used with chemical-selective fatsuppression. Parameters for these scans were: TR 2 R-R intervals, echotrain length 16, bandwidth 62.5 kHz, field of view 14 cm, slicethickness 3 mm, acquired matrix 256×256, reconstructed matrix 512×512,with one excitation. Slice levels were centered at the carotidbifurcation in each patient. Two board-certified radiologists blinded toall other information determined the presence or absence of intra-plaquelipid, based on the loss of signal between the proton density weightedimages and the fat-suppressed moderately-T2 weighted fast spin echoimages with iso-intense signal on 3D time-of-flight imaging.¹⁵ Thepercent diameter stenosis was calculated as the difference betweenreference and stenotic diameters on 3D time-of-flight axial images,divided by the reference diameter and multiplied by 100. The images wereread independently off-line. Inter-observer variability was <10%.

Baseline plasma soluble CD40 ligand concentrations were measured byELISA as previously described.¹⁰ In brief, 1:5 diluted plasma sampleswere applied in triplicate to 96-well plates precoated with mouseanti-human CD40 ligand antibody and mixed (1:2) with ahorseradish-peroxidase-labeled secondary mouse anti-human CD40 ligandantibody for two hours. Plates were then washed and antibody bindingdetermined by colorimetry using 3,3′-5,5′-tetramethyl benzidinesubstrate. Absorbance was read at 650 nm and plasma concentrations ofsoluble CD40 ligand were determined by comparison with serial dilutionsof recombinant human CD40 ligand. Intra-assay variation among thetriplicates for all samples was less than 10% and inter assayvariability was 7.4%.

We divided the study participants into two groups, those with evidenceof intra-plaque lipid on carotid MRI, and those without evidence ofintra-plaque lipid. Median levels of soluble CD40 ligand were computedand compared between the two groups using Wilcoxon's ranked sum test.Relative risks of having intra-plaque lipid associated with elevatedsoluble CD40 ligand levels were computed by use of logistic regressionmodels that divided the study sample according to the median level ofsoluble CD40 ligand among those without evidence of intra-plaque lipid.

Results

Of the 49 patients enrolled, carotid MRI images could not be obtainedfor 3 patients due to claustrophobia. We utilized the observations foranalyses of the remaining 46 patients. The baseline clinicalcharacteristics of the patients (Table IV) revealed that there was ahigh prevalence of a history of hypertension, diabetes, andhypercholesterolemia in the overall study cohort. Thirteen of the 46patients (28.3%) had a prior history of transient ischemic attack (TIA)(n=8) or stroke (n=6); one patient had suffered both TIA and stroke. Theremaining 33 patients (71.7%) were asymptomatic.

Fourteen patients had evidence of intra-plaque lipid and 32 did not.Patients with evidence of intra-plaque lipid more likely had diabetes(p=0.02) than those patients without evidence of intra-plaque lipid.There was also a trend towards an increased proportion of women (p=0.1),patients with a history of hypertension (p=0.16), and current smokers(p=0.13) in the group with intra-plaque lipid. Mean percent carotiddiameter stenosis (58±20 vs 56±24) did not differ between those with andwithout evidence of intra-plaque lipid.

Subjects with intra-plaque lipid had higher baseline levels of solubleCD40 ligand than among those without lipid accumulations (median 2.54ng/ml [interquartile range (IQR) 1.85-3.52] vs median 1.58 ng/ml [IQR1.21-2.39]; p=0.02). In contrast, soluble CD40 ligand levels did notcorrelate with percent diameter stenosis (r=−0.19; p=0.21). The relativerisk for intra-plaque lipid associated with soluble CD40 ligand levelsabove the median was 6.0 (95% confidence interval 1.15-31.23; p=0.03).The magnitude of this predictive effect did not substantially changewhen analyzed by a multivariable model controlling for the effects ofgender, diabetes, hypertension, current smoking, percent stenosis, andratio of total cholesterol to high density lipoprotein cholesterol(relative risk 5.12, 95% confidence interval 0.78-33.73; p=0.09). TABLEIV Baseline Clinical Characteristics of the Study Population.Intra-plaque P value Total No Intra-plaque lipid (Group 1 Cohort lipidGroup 1 Group 2 vs Group (n = 46) (n = 32) (n = 14) 2) Age (years) 70.5± 8.1 71.0 ± 7.9 69.4 ± 8.8 0.5 mean ± SD Male Gender 31/46 24/32 (75%) 7/14 (50%) 0.10 (67.4%) History of 15/46  7/32 (21.9%)  8/14 (57.1%)0.02 Diabetes (32.6%) History of 37/46 24/32 (75%) 13/14 (92.9%) 0.16Hypertension (80.4%) Current  5/46  2/32 (6.3%)  3/14 (21.4%) 0.13Smoker (10.9%) History of High 34/46 23/32 (71.9%) 11/14 (78.6%) 0.6Cholesterol (73.9%) Prior TIA or 13/46  8/32 (25%)  5/14 (35.7%) 0.46Stroke (28.3%) Statin use 31/46 20/32 (62.5%) 11/14 (78.6%) 0.28 (67.4%)Percent 57% ± 23 56% ± 24 58% ± 20 0.6 diameter stenosis Soluble CD401.89 1.58 2.54 0.02 ligand (ng/ml) [1.35- [1.21-2.39] [1.85-3.52] median[inter- 2.64] quartile range]TIA = transient ischemic attackDiscussion

As described elsewhere herein (Example 1), baseline plasma levels ofsoluble CD40 ligand prospectively predict cardiovascular events amongapparently healthy women. The current data provide novel insight intothe mechanism through which elevated levels of soluble CD40 ligand mayreflect future cardiovascular risk in humans. We found an associationbetween elevated plasma levels of soluble CD40 ligand and carotidplaques with features of high risk without relation to the severity ofstenosis. These data agree with evidence from studies showing thatinterruption of CD40 signaling reduced the size and lipid content ofaortic lesions in atherosclerosis-prone mice.⁹

Previous work has found that high-resolution carotid MRI, using asimilar phased array carotid coil, can accurately predict histologicalfindings of lipid pool following carotid endarterectomy ¹⁵; our MRIprotocol relies more on T2-weighted protocols, which other studies haveshown can accurately distinguish lipid pool.^(12,13,16) The predictiveeffect of soluble CD40 ligand persisted after adjustment for traditionalcardiovascular risk factors, although the confidence intervals did widenin the adjusted analysis, as might be expected given that CD40 ligationappears to represent a common causal pathway in lipid pool formation.

In conclusion, we believe that this study establishes a link betweenplasma levels of CD40 ligand and intra-plaque lipid, which representsone potentially important marker of plaque vulnerability.

REFERENCES INCORPORATED IN EXAMPLE 3

1. Davies M J. Circulation 1996; 94:2013-20.

2. Libby P Circulation 1995; 91:2844-50.

3. Karmann K, et al. Proc Natl Acad Sci USA 1995; 92:4342-6.

4. Kornbluth R S, et al. Proc Natl Acad Sci USA 1998; 95:5205-10.

5. Mach F, et al. J Clin Invest 1999; 104:1041-50.

6. Denger S, et al. Atherosclerosis 1999; 144:15-23.

7. Mach F, et al. Circulation 1997; 96:396-9.

8. Schonbeck U, Am J Pathol 2000; 156:7-14.

9. Schonbeck U, J Exp Med 1999; 189:843-53.

10. Schonbeck U, Circulation2001; 104:2266-8.

11. Fayad Z A, Fuster V. Circ Res 2001; 89:305-16.

12. Shinnar M, et al. Arterioscler Thromb Vasc Biol 1999; 19:2756-61.

13. Serfaty J M, et al. Radiology 2001; 219:403-10.

14. Hatsukami T S, et al. Circulation 2000; 102:959-64.

15. Yuan C, et al. Circulation 2001; 104:2051-6.

16. Toussaint J F, et al. Arterioscler Thromb Vasc Biol 1995;15:1533-42.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

1.-11. (canceled)
 12. A method for characterizing an individual's riskprofile of developing a future cardiovascular disorder associated withatherosclerotic disease, comprising: obtaining a level of sCD40L in theindividual, comparing the sCD40L level to a first predetermined value toestablish a first risk value, obtaining a level of a cholesterol orC-Reactive Protein (CRP) in the individual, comparing the level of thecholesterol or C-Reactive Protein (CRP) to a second predetermined valueto establish a second risk value, and characterizing the individual'srisk profile of developing the cardiovascular disorder based upon thecombination of the first risk value and the second risk value, whereinthe combination of the first risk value and second risk valueestablishes a third risk value different from said first and second riskvalues.
 13. The method of claim 12, wherein said individual is anapparently healthy, non-smoking individual. 14.-17. (canceled)
 18. Themethod of claim 12, wherein the cardiovascular disorder is stroke. 19.The method of claim 12, wherein the cardiovascular disorder is nonfatalmyocardial infarction.
 20. (canceled)
 21. A method for evaluating thelikelihood that an individual will benefit from treatment with an agentfor reducing the risk of a cardiovascular disorder associated withatherosclerotic disease, the agent selected from the group consisting ofanti-inflammatory agents, anti-thrombotic agents, anti-platelet agents,fibrinolytic agents, lipid reducing agents, direct thrombin inhibitors,and glycoprotein II b/IIIa receptor inhibitors comprising: obtaining alevel of sCD40L in the individual, and comparing the level of sCD40L toa predetermined value, wherein the level of sCD40L in comparison to thepredetermined value is indicative of whether the individual will benefitfrom treatment with said agents, and characterizing whether theindividual is likely to benefit from said treatment based upon saidcomparison.
 22. (canceled)
 23. The method of claim 21, wherein saidindividual is an apparently healthy; non-smoking individual. 24.-27.(canceled)
 28. The method of claim 21, wherein the cardiovasculardisorder is stroke.
 29. The method of claim 21, wherein thecardiovascular disorder is myocardial infarction.
 30. A method fortreating a subject to reduce the risk of a cardiovascular disorder,comprising: selecting and administering to a subject who is known tohave an above-normal level of sCD40L an agent for reducing the risk ofthe cardiovascular disorder in an amount effective to lower the risk ofthe subject developing a future cardiovascular disorder, wherein theagent is an anti-inflammatory agent, an antithrombotic agent, ananti-platelet agent, a fibrinolytic agent, a lipid reducing agent, adirect thrombin inhibitor, a glycoprotein IIb/IIIa receptor inhibitor,an agent that binds to cellular adhesion molecules and inhibits theability of white blood cells to attach to such molecules, a calciumchannel blocker, a beta-adrenergic receptor blocker, a cyclooxygenase-2inhibitor, or an angiotensin system inhibitor.
 31. The method of claim30, wherein the subject is otherwise free of symptoms calling fortreatment with the agent.
 32. The method of claim 30, wherein thesubject is apparently healthy and the subject does not have an elevatedrisk of an adverse cardiovascular event.
 33. The method of claim 30,wherein the subject is nonhyperlipidemic.
 34. The method of claim 30,wherein the agent is a non-aspirin, anti-inflammatory agent. 35.-38.(canceled)
 39. The method of claim 30, wherein the agent is a lipidreducing agent.
 40. The method of claim 39, wherein the lipid reducingagent is gemfibrozil, cholystyramine, colestipol, nicotinic acid,probucol lovastatin, fluvastatin, simvastatin, atorvastatin,pravastatin, or cerivastatin. 41.-70. (canceled)
 71. A method forreducing sCD40L levels in a subject to lower the risk of an adversecardiovascular disorder, comprising: selecting and administering to asubject having elevated levels of sCD40L an agent that reduces sCD40Llevels in an amount effective to reduce the sCD40L levels in thesubject.
 72. The method of claim 71, wherein the agent is a lipidlowering agent.
 73. The method of claim 71, wherein the subject isapparently healthy.
 74. The method of claim 71, wherein the subject isnot otherwise at an elevated risk of having an adverse cardiovascularevent.
 75. The method of claim 71, wherein the subject has elevatedC-Reactive Protein (CRP) levels.
 76. The method of claim 71, wherein thesubject is otherwise free of indications calling for treatment with alipid reducing agent.